過氧化物酶體增長因子活化受體拮抗劑和激動劑的篩選方法
2023-10-22 06:25:37 5
專利名稱：過氧化物酶體增長因子活化受體拮抗劑和激動劑的篩選方法
技術領域：
本發明涉及利用分子和細胞生物學技術篩選PPAR拮抗劑和激動劑的方法，具體涉及利用酵母雙雜交技術，通過測定α-半乳糖苷酶的活性大小篩選PPAR拮抗劑和激動劑的方法。
背景技術：
近年來，在我國隨著人們生活水平質量的不斷提高，飲食結構及生活方式的變化，患有血脂代謝異常的人數在逐年升高，由此而引發的高血壓病、高脂血症、冠心病、糖尿病、肥胖症等「富裕性疾病」的發病率亦明顯上升，同時，這類疾病還是威脅中老年人健康的第一大殺手，已經引起了全社會的高度重視。有資料顯示，目前，高脂血症、糖尿病、肥胖症等相關性十分密切的病症，其相加總發病率已達10％。我國90年代初期與80年代相比，人群血脂水平明顯增加，尤其在北方大城市，約30～40％的人患有不同程度的超過邊緣性標準的血脂和血糖代謝異常。因此，在全社會範圍內重視、控制這類疾病已勢在必行。
近十年來的研究表明，核受體PPAR(Peroxisome Proliferator-activated Receptors)又稱過氧化物酶體增長因子活化受體，是一類由配體激活的核轉錄因子，屬II型核受體超家族成員，能被脂肪酸以及外源性過氧化物酶體增殖劑(Peroxisome Proliferator，PP)激活。在兩棲類、嚙齒類動物及人類等PPARs均有3種亞型，即PPARα、PPARβ(亦稱PPARδ或NUC1)和PPARγ。人類PPARα、β和γ分別含有468、441和479個胺基酸殘基。作為甾體激素受體超家族成員之一，PPAR共有6個區域(A-F)、4個功能結構域。位於受體分子中部的C區是DNA結合區(DBD)；羧基端的E/F區是配基結合區(LBD)，在激素信號轉換成轉錄激活信號過程中發揮重要作用；氨基端的A/B是調節區，PPARγ的活化可被MAPK介導的A/B區的一個絲氨酸殘基的磷酸化而降低，而PPARα的磷酸化可正調節受體-配體的親和力。活化的PPAR與視黃酸類受體(RXR)形成異二聚體，然後與所調節基因的啟動子上遊的過氧化物酶體增殖物反應元件(PPRE)結合而發揮轉錄調控作用。同時，活化的PPAR能同一系列轉錄共激活因子(如CBP SRC-1；RIP140；TIF1；TIF2；SUG1；P300)結合，從而激活轉錄。PPAR介導的效應主要包括脂類代謝、免疫反應等，因此，動脈粥樣硬化、肥胖、糖尿病、腫瘤以及炎症性疾病等的發生發展與PPAR有密切關係。
PPAR的不同亞型具有不同的生物學作用，PPARα在肝臟脂類代謝中發揮重要作用，PPARβ在脂肪細胞分化中的作用還存在著爭議，而PPARγ參與脂肪形成和免疫反應，與脂肪細胞分化、肥胖及胰島素抵抗關係密切，被認為是治療糖尿病、肥胖症、壞脂血症、炎症、高血壓和癌症藥物設計的重要靶標分子，成為近年來研究熱點。
對於PPARγ激動劑的研究主要集中在其在治療II型糖尿病方面的應用。胰島素增敏劑噻唑烷二酮類藥物(TZDs)作為PPARγ的一類激動劑可以明顯降低白色脂肪組織(WAT)中甘油三酯的含量，同時還可降低血漿中的葡萄糖，脂質和II型糖尿病實驗動物的胰島素水平。三類TZD類藥物羅格列酮rosiglitazone(Rezulin)，皮格列酮piogitazone(ACTOS)，和曲格列酮Troglitazone(Avandia)已被FDA認證通過，作為治療II型糖尿病的藥物已經在市場上銷售。至於PPARγ拮抗劑，理論上可減少脂肪、防治肥胖，但臨床尚未應用。
目前國內在PPARγ激動劑和拮抗劑的高通量篩選上大都局限於蛋白質水平的小分子化合物的篩選，如SPR技術，而細胞水平的篩選則基本依賴於哺乳動物細胞，成本較高，操作也相對煩瑣。酵母雙雜交技術是一項採用報告基因來檢測蛋白質與蛋白質間相互作用的技術(美國專利No.5,283,173；Proc.Natl.Acad.Sci.，USA，vol.88，pp.9578-9582，1991)。許多轉錄因子可以分為DNA結合域(DNA binding domain，DNA-BD)和轉錄激活域(transcriptional activation domain，AD)，而只有DNA-BD和AD這兩個結構域共存的情況下，才能激活下遊基因的表達。酵母雙雜交系統中，將待檢測的蛋白X、Y分別與DNA-BD、AD表達為融合蛋白DNA-BD-X、AD-Y，只有在X、Y有相互作用的情況下，DNA-BD、AD方形成完整的複合物，激活下遊報告基因的表達。目前通常選用LacZ作為酵母雙雜交的報告基因，然而β-半乳糖苷酶是一種胞內酶，因此酵母細胞的裂解徹底與否會對酶活的測定造成直接影響，且操作相對複雜。
發明內容
本發明的目的在於提供一種利用酵母雙雜交系統來篩選PPAR拮抗劑和激動劑的新方法。
本發明的另一個目的是提供一種通過測定α-半乳糖苷酶的活性大小，來方便而快捷的檢測小分子化合物對PPAR與共激活因子間相互作用影響的方法。
本發明的再一個目的是提供用於本發明方法篩選PPAR拮抗劑和激動劑的酵母篩選系統。該系統包括(1)小鼠共激活因子CBP和酵母轉錄因子GAL4-AD融合蛋白表達質粒pGADT7-CBP(Escherichia E.coli DH5α)保藏號為CGMCC No.1076，保藏日期2003年12月15日，地址北京中關村中國微生物菌種保藏管理委員會普通微生物中心，郵編100080；(2)含人PPARγLBD和酵母轉錄因子GAL4-BD融合蛋白表達質粒pGBKT7-PPARγLBD(Escherichia E.coli DH5α)保藏號為CGMCC No.1077，保藏日期2003年12月15日，地址北京中關村中國微生物菌種保藏管理委員會普通微生物中心，郵編100080；(3)宿主酵母AH109。
本發明採用了酵母雙雜交這一有利的研究蛋白質與蛋白質之間相互作用的技術，在PPARs與共激活因子的結合是配體依賴性前提下，實現了PPAR的激動劑和拮抗劑的篩選。本發明採用GAL4轉錄因子，其DNA-BD、AD區域則已分別構建在酵母表達質粒pGBKT7與pGADT7中。本發明採用酵母AH109基因組自帶的MEL1基因作為雙雜交的報告基因，通過測定其分泌型的產物α-半乳糖苷酶的活性來檢測小分子化合物對PPARγ與其共激活因子相互作用程度的影響。
本發明利用酵母雙雜交系統來篩選PPAR拮抗劑與激動劑的方法由下列步驟組成1、CBP和酵母GAL4AD融合蛋白(pGADT7-CBP)的質粒構建；本步驟是藉助細胞分子生物學方法，選用pGADT7載體構建CBP和酵母GAL4AD融合蛋白的質粒(pGADT7-CBP)。
(1)用PCR技術從CMV-mCBP中獲得CBP(1-464aa)的DNA片段引物FW5』AACATATGATGGCCGAGAACTTGCTGGACG 3』RV5』AAGGATCCCTGTTGCCCTGCACCAACAG 3』95℃變性8min，開始循環95℃ 1min，65℃ 1min，72℃ 1min30s，重複30循環，72℃ 10min。
採用瓊脂糖凝膠電泳鑑定PCR產物，回收DNA。
(2)將CBP(1-464aa)的PCR產物克隆到pGADT7載體上CBP(1-464aa)的PCR產物和pGADT7分別用Nde I和BamHI酶切；用試劑盒回收酶切產物，其中回收CBP(1-464aa)的PCR酶切產物近1400bp，pGADT7酶切產物近8.0kb。用T4DNA連接酶連接；連接產物轉化入感受態細胞DH5α；通過酶切鑑定挑選正確的克隆。
2、PPARγLBD和酵母GAL4BD融合蛋白(pGBKT7-PPARγLBD)的質粒構建；本步驟是藉助細胞分子生物學方法，選用pGBKT7載體構建PPARγLBD和酵母GAL4DNA-BD融合蛋白的質粒(pGBKT7-PPARγLBD)。
(1)利用PCR技術從pCDNA3.1-PPARγ中獲得PPARγLBD(193aa-475aa)的DNA片段引物FW5』AACATATGGCGGAGATCTCCAGT 3』RV5』AAGTCGACCTAGTACAAGTCCTT 3』95℃變性5min，開始循環95℃ 30s，60℃ 30s，72℃ 1min，重複30循環，72℃10min。
採用瓊脂糖凝膠電泳鑑定PCR產物，回收DNA。
(2)將PPARγLBD的PCR產物克隆到pGBKT7載體上PPARγLBD的PCR產物和pGBKT7分別用Nde I和Sal I酶切；用試劑盒回收酶切產物，其中回收PPARγLBD的PCR酶切產物近850bp，pGBKT7酶切產物近7.3kb；用T4DNA連接酶連接；連接產物轉化入感受態細胞DH5α；通過酶切鑑定挑選正確的克隆。
3、將質粒pGBKT7-PPARγLBD和pGADT7-CBP依次轉入酵母細胞AH109，得到酵母PPARγLBD-CBP_AH109(Saccharomyces cerevisiae AH109)保藏號為CGMCC No.1075，保藏日期2003年12月15日，地址北京中關村中國微生物菌種保藏管理委員會普通微生物中心，郵編100080採用LiAc轉化法。先將pGBKT7-PPARγLBD質粒轉入酵母細胞AH109，用色氨酸缺陷(T-)的培養基篩選陽性克隆，然後將pGADT7-CBP質粒轉入含有pGBKT7-PPARγLBD質粒的酵母中，用色氨酸，亮氨酸雙缺陷(T-L-)的培養基篩選陽性克隆，得到同時含有pGADT7-CBP質粒和pGBKT7-PPARγLBD質粒的酵母細胞。
4、通過測定α-半乳糖苷酶的活性篩選化合物在得到的含pGADT7-CBP和pGBKT7-PPARγLBD雙質粒的陽性酵母細胞中分別加入陽性對照物、陰性對照物及待篩選的化合物，孵育後測定α-半乳糖苷酶的活性，並計算其激動率，根據各個待篩選藥物的激動率的比較，篩選出PPAR拮抗劑和激動劑。
本發明提供了利用酵母雙雜交系統定量檢測小分子化合物對蛋白質相互作用強度影響方法採用酵母基因組自帶的MEL1基因作為雙雜交的報告基因，通過測定其分泌型的產物α-半乳糖苷酶的活性來檢測小分子化合物對PPARγ與其共激活因子相互作用程度的影響。
本發明利用載體pGADT7構建的pGADT7-CBP質粒的核苷酸序列如下TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAA
ATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGatggccgagaacttgctggacggaccgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgacttggaaaatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaaacataaacaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgtgcaacagggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctgaaccaaggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatccacaagcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttcaaccagacccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatggatctcttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctggcggagaccttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatgggaatgactggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagttagccagcaaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtcccagttgcaaacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacagcagcagctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacactgtcgaaccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaaatcatctctcattggaagaactgcacacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaaccatcctgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacagGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACTCCTCTAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTATTCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGA
AAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGACTCTAGCTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGATCCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG
CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCT
AGCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTGCTpGADT7-CBP質粒表達的胺基酸序列為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 SerLys 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 MetMet Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys ArgAla Lys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe AspLeu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser GlyAsn Leu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser GlySer Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly GlyGln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser ProLeu Asn Gln Gly Asp Ser Ser Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly ProThr Pro Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu Val Thr Ser SerPro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met Asn Ala Asn Phe Asn Gln Thr His ProGly Leu Leu Asn Ser Asn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala GlnVal Met Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro AlaPro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro GlnMet 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 ThrGly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr AspIle Lys Asn Thr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile ValPro Thr Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln GlnLeu Val Leu Leu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val ArgAla Cys Ser Leu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys GlnAla Pro Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys AsnCys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu Lys Asn Ala Ser Asp Lys Arg Asn GlnGln Thr Ile Leu Gly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly GlnGlnGly Ser Ile Glu Leu Glu Leu Gln Met Asn Arg Arg Tyr***本發明利用載體pGBKT7構建的pGBKT7-PPARγLBD質粒，具有如下核苷酸序列CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaacatttgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaatcaccattcgttatctatgacatgaattccttaatgatgggagaagataaaatcaagttcaaacacatcacccccctgcaggagcagagcaaagaggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagatcacagagtatgccaaaagcattcctggttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatga
ataaagatggggttctcatatccgagggccaaggcttcatgacaagggagtttctaaagagcctgcgaaagccttttggtgactttatggagcccaagtttgagtttgctgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtggagaccgcccaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctgaaccaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctactgcaggtgatcaagaagacggagacagacatgagtcttcacccgctcctgcaggagatctacaaggacttgtactagGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGG
CGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAAC
CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATpGBKT7-PPARγLBD質粒表達的胺基酸序列如下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 Arg
Gln 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 MetAla Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala AspLeu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr Lys Ala LysAla Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn SerLeu Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys GluVal Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr GluTyr Ala Lys Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys TyrGly Val His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile SerGlu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp PheMet Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp LeuAla Ile Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile GluAsp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu SerSer Gln Leu Phe Ala Lys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His ValGln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu IleTyr Lys Asp Leu Tyr ***上述兩個質粒的核苷酸序列中帶下劃線的鹼基表示的是克隆該基因時所用的酶切位點；加粗的鹼基序列表示融合蛋白的序列；ATG和TGA也加粗表示起始密碼子和終止密碼子；小寫加粗的鹼基序列表示插入基因的序列；tag表示終止密碼子。
上述兩個質粒所表達的胺基酸序列中加黑的胺基酸序列表示的是融合蛋白的胺基酸序列；加黑並有下劃線的胺基酸序列表示的是插入基因的胺基酸序列；***表示終止。
圖1為pGADT7-CBP質粒構建策略圖。
圖2為CBP(1-464aa)PCR產物瓊脂糖電泳鑑定圖譜，圖譜左列為CBP(1-464aa)PCR產物電泳條帶，該條帶大小為1400bp，右列為100bp DNA marker。
圖3為pGADT7-CBP質粒酶切鑑定結果圖，其中0號為1kb DNA Marker；1號-9號為轉化平板上挑出的克隆。
圖4為pGADT7-CBP測序結果圖。
圖5為PGBKT7-PPARγLBD質粒構建策略圖。
圖6為PPARγLBD(193aa-475aa)PCR產物瓊脂糖電泳鑑定圖譜，圖譜左列為DNA長度標記，右列為PPARγLBD(193aa-475aa)PCR產物電泳條帶，該條帶大小為850bp。
圖7為PGBKT7-PPARγLBD質粒酶切鑑定結果，圖譜左列為轉化平板上挑出的克隆8號，右列為1kb DNA標記。
圖8為pGBKT7-PPARγLBD測序結果圖。
圖9為質粒pGBKT7-PPARγLBD轉化結果的PCR鑑定圖，其中1為100bp的DNAladder，2-4依次為所挑取的1-3號克隆，5為陽性對照(pGBKT7-PPARγLBD為模板)。
圖10為質粒pGADT7-CBP轉化結果的PCR鑑定圖，其中1為1kb的DNA標記，2為陰性對照(H2O為模板)，3為陽性對照(pGADT7-CBP為模板)，4-5為所挑取的1-2號克隆。
圖11為羅格列酮對CBP與PPARγ相互作用影響圖，其中1表示酵母菌株P1(含質粒pGBKT7-PPARγLBD)、2表示加入二甲亞碸後的酵母菌株P1、3表示加入羅格列酮(溶於二甲亞碸)後的酵母菌株P1、4表示酵母菌株P1C2(含雙質粒pGBKT7-PPARγLBD和pGADT7-CBP)、5表示加入二甲亞碸後的酵母菌株P1C2、6表示加入羅格列酮(溶於二甲亞碸)的酵母菌株P1C2。
圖12為藥物作用時間對檢測靈敏度的影響圖。
圖13為羅格列酮EC50的測定結果圖。
圖14為化合物篩選結果圖。
有益效果1、利用本發明可進行PPARγ拮抗劑和激動劑的高通量篩選，本發明採用酵母基因組自帶的MEL1基因作為雙雜交的報告基因，通過測定其分泌型的產物α-半乳糖苷酶的活性來檢測小分子化合物對PPARγ與其共激活因子相互作用程度的影響，無需裂解酵母細胞，是一種非常方便快捷且準確的方法，加之酵母細胞的易操作性有利於實現真核水平的高通量篩選。
2、利用本發明，首次在酵母水平進行了已知PPARγ激動劑-羅格列酮EC50的測定。
3、利用本發明可實現PPARγ拮抗劑和激動劑的高通量篩選，為藥物的結構修飾和天然先導化合物的發現提供實驗基礎。
具體實施例方式
下面結合具體實施例對本發明作進一步闡述，但不限制本發明。
下述實施例中，所用的試驗材料及其來源包括酵母菌株AH109(基因型為MATa，trp1-901，leu2-3，112，ura3-52，his3-200，gal4，gal80□...LYS2GAL1UAS-GAL1TATA-HIS3，MEL1UAS-MEL1TATA-MMEL1UAS-MEL1TATA-MEL1，GAL2UAS-GAL2TATA-ADE2，URA3MEL1UAS-MEL1TATA-lacZ)、酵母表達質粒pGBKT7和pGADT7為中科院上海生命科學學院龔毅教授惠贈；模板CMV-mCBP由M.G.Rosenfeld提供；模板pCDNA3.1-PPARγ由成都地奧公司提供；限制性內切酶、DNA聚合酶、連接酶、dNTP等均購自TaKaRa公司；10×buffer隨pyrobestTMDNA聚合酶一起購自TaKaRa公司；膠回收試劑盒購自華舜公司；
氨苄西林、卡那黴素、LiAc、PEG 3500、PNP-α-Gal、各培養基的配製原料均購自sigma公司；SS-carrier DNA購自Strategene；DNA marker購自鼎國生物公司；引物合成與DNA測序由上海博亞公司完成；96孔板購自Nunclon公司。
下述實施例中常規的基因操作參照有關文獻，其中限制性酶切方法參照分子克隆第二版P259-P262；用T4DNA連接酶連接方法參照分子克隆第二版P282-P283；連接產物轉化入感受態細胞DH5α方法參照分子克隆第二版P55-P56；用酶切鑑定方法參照分子克隆第二版P259-P262；瓊脂糖凝膠電泳鑑定PCR產物方法參照分子克隆第二版P680-P682；膠回收按照華舜公司的試劑盒說明書；質粒提取方法參照C.Hoffman andF.Winston，Gene 57267；1987；LB培養基的配製方法參照分子克隆第二版P908；酵母各培養基的配製方法參照Yeast Protocol Handbook(CLONTECH)。
實施例1 CBP和酵母GAL4AD融合蛋白(pGADT7-CBP)的質粒構建(一)試驗方法1)以CMV-mCBP為模板，用PCR技術複製擴增獲得CBP(1-464aa)的DNA片段(基因序列依據S66385(GENEBANK))引物設計FW5』AACATATGATGGCCGAGAACTTGCTGGACG 3』RV5』AAGGATCCCTGTTGCCCTGCACCAACAG 3』PCR擴增反應(100μl)CMV-mCBP模板2μl；10×buffer10μl；dNTP(2.5mM)8μl；FW4μl；RV4μl；pyrobestTMDNA聚合酶1μl；水71μl。
反應條件為95℃變性8min，開始循環95℃ 1min，65℃ 1min，72℃ 1min30s，重複30循環，72℃ 10min。
採用瓊脂糖凝膠電泳鑑定PCR產物，回收DNA。
2)將CBP(1-464aa)的PCR產物克隆到pGADT7載體上CBP(1-464aa)的PCR產物和pGADT7分別用Nde I和BamHI酶切；用試劑盒方法回收酶切產物，其中回收CBP(1-464aa)的PCR酶切產物近1400bp；pGADT7酶切產物近8.0kb；用T4DNA連接酶連接；連接產物轉化入感受態細胞DH5α；塗布在含氨苄西林(100mg/l)的平板上；用酶切鑑定挑選正確的克隆。
(二)試驗結果1、採用瓊脂糖凝膠電泳鑑定PCR產物的結果見圖2。
2、酶切鑑定結果在載體上1480bp，2280bp處有兩個Hind III的酶切位點，1480bp-2280bp間存在著多克隆位點，CBP就克隆在這個區域內，因而正確的克隆應切出約2200bp和7.2kb左右的片段。如圖3所示，經進一步酶切鑑定結果顯示確實有正確的克隆(5號和9號)。
3、DNA測序報告pGADT7-CBP表達載體測序報告如圖4所示，pGADT7-CBP DNA測序結果和CBP(1-464aa)原DNA序列比較，原序列1131處是一個未知鹼基N，5號在該處測出的是A，但這個位置是密碼子的第三位，所以無論是AGCT哪種，都不影響胺基酸編碼。
實施例2 PPARγLBD和酵母GAL4BD融合蛋白(pGBKT7-PPARγLBD)的構建(一)試驗方法以pCDNA3.1-PPARγ為模板，用PCR技術複製擴增獲得PPARγLBD(193aa-475aa)的DNA片段(基因序列依據NM005037(GENEBANK))引物設計FW5』AACATATGGCGGAGATCTCCAGT 3』RV5』AAGTCGACCTAGTACAAGTCCTT 3』PCR擴增反應(100μl)pCDNA3.1-PPARγ模板2μl；10×buffer10μl；dNTP(2.5mM)8μl；FW4μl；RV4μl；pyrobest聚合酶1μl；水71μl。
反應條件為95℃變性5min，開始循環95℃ 30s，60℃ 30s，72℃ 1min，重複30循環，72℃ 10min。
採用瓊脂糖凝膠電泳鑑定PCR產物，回收DNA。
2)將PPARγLBD的PCR產物克隆到pGBKT7載體上PPARγLBD的PCR產物和pGBKT7分別用Nde I和Sal I酶切；用試劑盒方法回收酶切產物，其中回收PPARγLBD的PCR酶切產物近850bp；pGBKT7酶切產物近7.3kb；用T4DNA連接酶連接；連接產物轉化入感受態細胞DH5α；塗布在含卡那黴素(50mg/l)的平板上用酶切鑑定，挑選正確的克隆。
(二)試驗結果1、採用瓊脂糖凝膠電泳鑑定PCR產物的結果見圖6。
2、酶切鑑定結果在載體上6313bp處有一個EcoRV的酶切位點，PPARγLBD基因內部16bp處也有一個EcoRV的酶切位點，因而正確的克隆應切出約2200bp和6kb左右的片段。如圖7所示，經進一步酶切鑑定結果顯示確實有正確的克隆(8號)。
3、DNA測序報告pGADT7-CBP表達載體測序報告如圖8所示，PPARγLBDDNA測序結果和PPARγLBD原DNA序列比較，結果完全一致。
實施例3 將pGADT7-CBP與pGBKT7-PPARγLBD分步轉入酵母細胞AH109中
(一)試驗方法1)按LiAC法將質粒pGBKT7-PPARγLBD轉入酵母細胞AH109接種酵母菌株AH109於約3ml YPDA中，30℃、250rpm培養16-18h(OD600約為1.4)；菌液按1∶5轉接於新鮮的YPDA中，30℃、200rpm培養5-7h(OD600至0.8～1.2)；取10ml菌液，4000rpm*5min離心收集酵母細胞；棄上清，1ml無菌水重懸，轉移入另一離心管中，再加入約10ml無菌水，混勻；4000rpm*5min再次離心收集酵母細胞；棄上清，1ml無菌水重懸，轉移入1.5ml eppendorf管中；4000rpm*5min再次離心收集酵母細胞，小心吸去上清；依次加入240μl PEG 3500(50％W/V)、36μl 1.0M LiAc(pH7.5)、5μl煮沸的SS-carrier DNA(10mg/ml)、60μl無菌水和10μl待轉化質粒。
劇烈振蕩1min；30℃靜置30min；42℃水浴靜置30min，每隔5min顛倒混勻一次；4000rpm*5min離心收集轉化的酵母細胞；吸去上清，1ml無菌水重懸，取200μl塗於塗布於色氨酸缺陷(以下簡稱為T-)的選擇平板上，30℃培養3-4d；2)鑑定攜帶pGBKT7-PPARγLBD質粒的陽性克隆以上T-選擇平板上長出的克隆中，挑取1-3號至T-液體培養基中，30℃、250rpm培養16-18h；收集酵母，提取質粒，PCR鑑定所提質粒引物序列FW5』GTAATACGACTCACTATAGGGCGA 3』RV5』TCCGAGTCACTTTAAAATTTGTAT 3』PCR擴增反應(20μl)模板(所提質粒)14μl；10×buffer2μl；dNTP(2.5mM)2μl；FW1μl；RV1μl；taq DNA polymerase0.5μl。
反應條件為95℃變性5min，開始循環95℃ 45s；50℃ 45s；72℃ 1min，重複35循環，72℃變性10min。
3)PCR鑑定得到擁有pGBKT7-PPARγLBD質粒的陽性克隆1，結果如圖9所示。
4)將質粒pGADT7-CBP轉入到上述1號克隆中接種1號克隆於約3ml T-液體培養基中，30℃、250rpm培養16-18h(OD600約為1.4)；菌液按1∶5轉接於新鮮的T-液體培養基中，30℃、200rpm培養5-7h(OD600至0.8～1.2)；
按以上所述步驟轉化1號克隆，最終塗布於色氨酸、亮氨酸雙缺陷(以下簡稱為T-L-)平板，30℃培養3-4d；5)PCR鑑定質粒pGADT7-CBP是否轉入1號克隆挑取T-L-平板上的1-3號克隆至T-L-液體培養基中，30℃、250rpm，培養16-18h後提質粒(方法同前)，PCR鑑定引物序列FW5』GTAATACGACTCACTATAGGGCGA 3』RV5』AGATGGTGCACGATGCACAGTT 3』PCR擴增反應(20ul)模板(所提質粒)14μl；10×buffer2μl；dNTP(2.5mM)2μl；FW1μl；RV1μl；taq DNA聚合酶0.5μl。
反應條件95℃變性5min，開始循環95℃ 45s；50℃ 45s；72℃ 1min，重複35循環；72℃變性10min6)鑑定得到擁有pGBKT7-PPARγLBDpGADT7-CBP兩個質粒的陽性克隆2，結果如圖10所示。
實施例4 篩選化合物1、羅格列酮(陽性化合物)對CBP與PPARγ相互作用影響的鑑定1)將擁有pGBKT7-PPARγLBD質粒的1號克隆(以下簡稱為P1)和擁有雙質粒pGBKT7-PPARγLBDpGADT7-CBP的2號克隆(以下簡稱P1C2)分別劃線於T-、T-L-平板，30℃培養3-4d後，取新鮮克隆(1-3周)，分別接種於2ml T-、T-L-液體培養基中，30℃、250rpm，過夜培養16-18h；2)分別用T-、T-L-液體培養基稀釋P1、P1C2過夜菌(至OD600約0.3)，按1∶100加入羅格列酮(自製合成，溶於二甲亞碸(DMSO)，10mM)或DMSO，實驗分組情況如下表
*每組均設三份平行。
30℃培養6h後，測定α-半乳糖苷酶活性(按照Yeast Protocol Handbook(CLONTECH)進行)混勻酵母培養液，取200μl測定OD 600(HITACHI，分光光度計U-2010)；另取200μl菌液於1.5ml eppendorf管中，10,000rpm*5min離心；
取16μl上清於96孔板中(T-、T-L-液體培養基分別用作空白對照)，每孔加入48μl分析緩衝液(100mM PNP-a-Gal水溶液與0.5M醋酸鈉，pH4.5按體積比1∶2混合)；30℃避光孵育30min；加入136μl 10X終止緩衝液(1M Na2CO3)，終止反應；測定410nm處的OD值(BIO-RAD酶標儀MODEL 550)。
α-半乳糖苷酶活性的計算公式為α-半乳糖苷酶活性[mIU/(ml×cell)]＝OD410×Vf×1,000/[(ε×b)×t×Vi×OD600]其中，t＝反應時間(分)Vf＝測值時的終體積(200ul)Vi＝加入酵母培養液上清的體積(16ul)OD600＝酵母培養液中酵母在600nm處的吸光度ε×b＝對-硝基苯酚在410nm處的摩爾吸光度×比色杯的底邊邊長(cm)＝10.5(ml/μmol)(對於200μl菌液而言)3)試驗結果，各試驗組的α-半乳糖苷酶活性檢測結果如圖11所示由圖11可見，加入羅格列酮能有效提高實驗菌株P1C2的α-半乳糖苷酶活性，作為溶劑的DMSO對酶活則基本無影響；而相同條件下對P1菌株進行α-半乳糖苷酶活性檢測，結果均為陰性。可見，羅格列酮作為一種已知的PPAR.γ激動劑，其與PPAR.γ的結合改變了PPAR.γ的構象，使得配體依賴性的PPAR.γ與CBP的結合得以增強，最終促進了GAL4依賴的MEL1基因表達其產物α-半乳糖苷酶。
2、藥物孵育時間對檢測靈敏性的影響由於目前尚無用α-半乳糖苷酶做為報告基因來定量檢測藥物效力，藉以篩選新的先導化合物的相關報導，而文獻報導的採用β-半乳糖苷酶做為酵母雙雜交中的報告基因進行藥物篩選時，有關加藥時間的確定也並不統一，從4-5h至過夜孵育不等，更無人做過加藥時間與靈敏度間相關性的有關工作。
接P1C2於T-L-液體培養基中，30℃、250rpm培養過夜，基於更靈敏地檢測，用T-L-液體培養基對過夜菌作不同程度的稀釋(按下表)，以便測酶活時，各組菌均能達到對數生長中期。
按1∶100加入羅格列酮(10mM)或DMSO，30℃孵育。在孵育2h、4h、6h、8h、10h、12h、14h時(每隔2h)取樣，按照試驗1所述的方法，測定各試驗組α-半乳糖苷酶活性。並計算α-半乳糖苷酶相對活性，其公式為
結果如圖12所示，延長藥物作用的時間可提高該系統檢測的靈敏度，12-14小時的孵育時間可使本系統靈敏度足以滿足藥物篩選的需要。
由以上實驗，可以看出，PPAR.γ、CBP間這種配體依賴性的相互作用在以上的實驗系統中靈敏穩定地被定量檢測與衡量，可用於篩選能充當PPAR.γ配體的天然化合物(激動劑或拮抗劑)。
3、羅格列酮(陽性化合物)EC50的測定接P1C2於T-L-液體培養基中，30℃、250rpm培養過夜，用T-L-液體培養基稀釋過夜菌至OD600約0.08後，以1∶100分別加入不同濃度的羅格列酮(10-9M、10-8M、10-7M、10-6M、10-5M、10-410-5M)或DMSO，30℃培養12h後，按照試驗1所述的方法，測定各試驗組α-半乳糖苷酶活性，並計算α-半乳糖苷酶相對活性。結果如圖13所示，羅格列酮對CBP與PPARγ間相互作用的促進作用是濃度依賴性的，並據此可得到其在該系統中EC50為1.3E-6。由此，羅格列酮在本系統中可作為陽性化合物來衡量待篩藥物的活性。
4、藥物篩選接P1C2於T-L-液體培養基中，30℃、250rpm培養過夜，T-L-液體培養基稀釋後，按1∶100分別加入羅格列酮(10mM)、DMSO以及待篩藥物(10mM)，30℃孵育12h後，進行α-半乳糖苷酶活性測定，並計算其激動率 本試驗中待篩藥物包括H001-H007為計算機藥物輔助設計所得，並經BIOCORE篩選；Z001-Z013為本實驗室所提取的中藥(初提物或純化合物)；結果如圖14所示H007、Z007表現出了強的激動效應，H001、H003、Z001、Z002也表現出了較好的激動效應；而H002、Z004則表現出了強的抑制效應。活性強的化合物可進一步測試其細胞或動物水平上的藥效，活性不強的則可通過計算機輔助設計進行結構修飾，以提高其活性。
序列表(1)一般信息(i)發明名稱PPAR拮抗劑和激動劑的篩選方法(ii)序列數目4(2)序列SEQ NO.1的信息(pGADT7-CBP的DNA序列)(i)序列特徵(A)長度9337bp(B)類型核苷酸(C)鏈性雙鏈(D)拓撲結構環狀(ii)分子類型DNA(iii)序列描述SEQ NO.1TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATAT℃CGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTAT
ACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGatggccgagaacttgctggacggaccgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgacttggaaaatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaaacataaacaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgtgcaacagggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctgaaccaaggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatccacaagcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttcaaccagacccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatggatctcttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctggcggagaccttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatgggaatgactggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagttagccagcaaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtcccagttgcaaacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacagcagcagctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacactgtcgaaccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaaatcatctctcattggaagaactgcacacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaaccatcctgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacagGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACTCCTCTAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTATTCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAACATATTC
CATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAA CGACGATCTTCTTAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGACTCTAGCTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGATCCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC
CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCT
TCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTGCT(3)序列SEQ ID NO.2的信息(pGADT7-CBP表達的胺基酸序列)(i)序列特徵(A)長度638AA(B)類型胺基酸(C)拓撲結構單鏈肽鏈(ii)分子類型蛋白質(iii)序列描述SEQ NO.2Met Asp Lys Ala Glu Leu Ile Pro Glu Pro Pro Lys Lys Lys Arg Lys Val Glu Leu Gly Thr Ala AlaAsn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser Leu Ser Phe Thr Phe Thr Asn Ser Ser AsnGly Pro Asn Leu Ile Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser Ser AsnVal His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp Asp Gly Asn Asn Ser LysPro Leu Ser Pro Gly Trp Thr Asp Gln Thr Ala Tyr Asn Ala Phe Gly Ile Thr Thr Gly MetPhe Asn Thr Thr Thr Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro ProAsn Pro Lys Lys Glu Ile Phe Asn Thr Thr His Tyr Arg Ala Ser Ala Ala Met Glu Tyr Pro Tyr AspVal Pro Asp Tyr Ala His MetMet Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg AlaLys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe Asp LeuGlu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser Gly AsnLeu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly SerSer Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly Gly GlnAla Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser Pro LeuAsn Gln Gly Asp Ser Ser Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly Pro Thr ProPro 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 LeuLeu Asn Ser Asn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln Val MetAsn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro Ala Pro AlaMet Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro Gln Met Ala GlyHis Ala Gly Leu Asn Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr ThrSer Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly Ala Thr Gly Val AsnPro Gln Leu Ala Ser LTs Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr Asp Ile LTs AsnThr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val Pro Thr GlnAla Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln Gln Leu Val LeuLeu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val Arg Ala Cys SerLeu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala Pro LysAla Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys Asn Cys Thr ArgHis Asp Cys Pro Val Cys Leu Pro Leu Lys Asn Ala Ser Asp Lys Arg Asn Gln Gln Thr Ile LeuGly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln GlnGly Ser Ile GluLeu Glu Leu Gln Met Asn Arg Arg Tyr ***(4)序列SEQ NO.3的信息(pGBKT7-PPARγLBD的DNA序列)(i)序列特徵(A)長度8128bp(B)類型核苷酸(C)鏈性雙鏈(D)拓撲結構環狀(ii)分子類型DNA(iii)序列描述SEQ NO.3CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCA
AAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaacatttgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaatcaccattcgttatctatgacatgaattccttaatgatgggagaagataaaatcaagttcaaacacatcacccccctgcaggagcagagcaaagaggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagatcacagagtatgccaaaagcattcctggttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatgaataaagatggggttctcatatccgagggccaaggcttcatgacaagggagtttctaaagagcctgcgaaagccttttggtgactttatggagcccaagtttgagtttgctgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtggagaccgcccaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctgaaccaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctactgcaggtgatcaagaagacggagacagacatgagtcttcacccgctcctgcaggagatctacaaggacttgtactagGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG
CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATA
GCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAA
CGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT(5)序列SEQ NO.4的信息(pGBKT7-PPARγLBD表達的胺基酸序列)(i)序列特徵(A)長度458AA(B)類型胺基酸(C)拓撲結構單鏈肽鏈(ii)分子類型蛋白質(iii)序列描述SEQ NO.4Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys Leu Lys Cys SerLys Glu Lys Pro Lys Cys Ala Cys Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro LysThr Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg LeuGlu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile Leu Lys Met Asp SerLeu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys Asp Ala ValThr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile SerAla Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu Thr Val Ser Pro Glu PheVal Ile Arg Leu Thr Ile Gly Arg Ala Ala Ile Met Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp LeuHis MetAla Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg Ala LeuAla Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala IleLeu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met MetGly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu Val Ala IleArg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala LysSer Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val HisGlu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile Ser Glu Gly GlnGly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu ProLys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe IleAla Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile Glu Asp Ile GlnAsp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln LeuPhe Ala Lys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln LeuLeu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu Ile Tyr LysAsp Leu Tyr ***
權利要求
1.一種PPAR拮抗劑和激動劑的篩選方法，其特徵在於利用酵母雙雜交系統，採用酵母AH109基因組自帶的MEL1基因作為雙雜交的報告基因，通過測定α-半乳糖苷酶的活性來檢測小分子化合物對PPAR與其共激活因子相互作用程度的影響，從而篩選PPAR拮抗劑和激動劑，包括如下步驟(1)CBP和酵母GAL4AD融合蛋白(pGADT7-CBP)的質粒構建；(2)PPARγLBD和酵母GAL4BD融合蛋白(pGBKT7-PPARγLBD)的質粒構建；(3)將質粒pGBKT7-PPARγLBD與質粒pGADT7-CBP轉入酵母細胞AH109，得到酵母PPARγLBD-CBP_AH109(保藏號為CGMCC No.1075，菌種於2003年12月15日保藏在北京中關村中國微生物菌種保藏管理委員會普通微生物中心)；(4)通過測定α-半乳糖苷酶的活性篩選化合物。
2.根據權利要求1所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於將質粒pGBKT7-PPARγLBD與質粒pGADT7-CBP依次轉入酵母細胞AH109的方法為採用LiAc轉化法，先將pGBKT7-PPARγLBD質粒轉入酵母細胞中，用色氨酸缺陷的培養基篩選陽性克隆，然後將pGADT7-CBP質粒轉入含有pGBKT7-PPARγLBD質粒的酵母中，用色氨酸，亮氨酸雙缺陷的培養基篩選陽性克隆，得到同時含有pGADT7-CBP質粒和pGBKT7-PPARγLBD質粒的酵母細胞。
3.根據權利要求1所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於通過測定α-半乳糖苷酶的活性篩選化合物的方法為在得到的含pGADT7-CBP和pGBKT7-PPARγLBD雙質粒的陽性酵母細胞中分別加入陽性對照物、陰性對照物及待篩選的化合物，孵育後，測定α-半乳糖苷酶的活性，並計算其激動率，根據各個待篩選藥物的激動率的比較，篩選出PPAR拮抗劑和激動劑。
4.根據權利要求3所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於通過測定α-半乳糖苷酶的活性篩選化合物時，藥物孵育時間為12-14小時。
5.根據利用權利要求1所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於該方法篩選PPAR拮抗劑和激動劑所利用的酵母篩選系統包括(1)小鼠共激活因子CBP和酵母轉錄因子GAL4-AD融合蛋白表達質粒pGADT7-CBP(菌種保藏號為CGMCC No.1076，菌種於2003年12月15日保藏在北京中關村中國微生物菌種保藏管理委員會普通微生物中心)；(2)含人PPARγLBD和酵母轉錄因子GAL4-BD融合蛋白表達質粒pGBKT7-PPARγLBD(菌種保藏號為CGMCC No.1077，菌種於2003年12月5日保藏在北京中關村中國微生物菌種保藏管理委員會普通微生物中心)；(3)宿主酵母AH109。
6.根據權利要求5所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於小鼠共激活因子CBP利酵母轉錄因子GAL4-AD融合蛋白表達質粒pGADT7-CBP具有如下序列(1)利用載體pGADT7構建的pGADT7-CBP質粒具有如下核苷酸序列TGCATGCCTGCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTTGCAAAGATGGATAAAGCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGatggccgagaacttgctggacggaccgcccaaccccaaacgagccaaactcagctcgcccggcttctccgcgaatgacaacacagattttggatcattgtttgacttggaaaatgaccttcctgatgagctgatccccaatggagaattaagccttttaaacagtgggaaccttgttccagatgctgcgtccaaacataaacaactgtcagagcttcttagaggaggcagcggctctagcatcaacccagggataggcaatgtgagtgccagcagccctgtgcaacagggccttggtggccaggctcaggggcagccgaacagtacaaacatggccagcttaggtgccatgggcaagagccctctgaaccaaggagactcatcaacacccaacctgcccaaacaggcagccagcacctctgggcccactccccctgcctcccaagcactgaatccacaagcacaaaagcaagtagggctggtgaccagtagtcctgccacatcacagactggacctgggatctgcatgaatgctaacttcaaccagacccacccaggccttctcaatagtaactctggccatagcttaatgaatcaggctcaacaagggcaagctcaagtcatgaatggatctcttggggctgctggaagaggaaggggagctggaatgccctaccctgctccagccatgcagggggccacaagcagtgtgctggcggagaccttgacacaggtttccccacaaatggctggccatgctggactaaatacagcacaggcaggaggcatgaccaagatgggaatgactggtaccacaagtccatttggacaaccctttagtcaaactggagggcagcagatgggagccactggagtgaacccccagttagccagcaaacagagcatggtcaatagtttacctgcttttcctacagatatcaagaatacttcagtcaccactgtgccaaatatgtcccagttgcaaacatcagtgggaattgtacccacacaagcaattgcaacaggccccacagcagaccctgaaaaacgcaaactgatacagcagcagctggttctactgcttcatgcccacaaatgtcagagacgagagcaagcaaatggagaggttcgagcctgttctctcccacactgtcgaaccatgaaaaacgttttgaatcacatgacacattgtcaggctcccaaagcctgccaagttgcccattgtgcatcttcacgacaaatcatctctcattggaagaactgcacaacgacatgactgtcctgtttgcctccctttgaaaaatgccagtgacaagcgaaaccaacaaaccatcctgggatctccagctagtggaattcaaaacacaattggttctgttggtgcagggcaacagGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTCAATTTCTTTCATTTATACATCGTTTTGCCTTCTTTTATGTAACTATACTCCTCTAAGTTTCAATCTTGGCCATGTAACCTCTGATCTATAGAATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTTGGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTATTCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCCGGTCGAAATTCCCCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAA AAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCGTCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTGGGGATCTCGACTCTAGCTAGAGGATCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGAGCGGCCGCGATCCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAACGCATTTAAGCATAAACACGCACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTATAGAATATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACAGTGCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTGCT(2)利用載體pGADT7構建的pGADT7-CBP質粒表達的胺基酸序列如下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 SerLys 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 HisMet Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys ArgAla Lys Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser Leu Phe AspLeu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly Glu Leu Ser Leu Leu Asn Ser GlyAsn Leu Val Pro Asp Ala Ala Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser GlySer Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln Gly Leu Gly GlyGln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala Ser Leu Gly Ala Met Gly Lys Ser ProLeu Asn Gln Gly Asp Ser Ser Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly ProThr Pro Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu Val Thr Ser SerPro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met Asn Ala Asn Phe Asn Gln Thr His ProGly Leu Leu Asn Ser Asn Ser Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala GlnVal Met Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro Tyr Pro AlaPro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu Thr Leu Thr Gln Val Ser Pro GlnMet Ala Gly His Ala Gly Leu Asn Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met ThrGly Thr Thr Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly Ala ThrGly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn Ser Leu Pro Ala Phe Pro Thr AspIle Lys Asn Thr Ser Val Thr Thr Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile ValPro Thr Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu Ile Gln Gln GlnLeu Val Leu Leu Leu His Ala His Lys Cys Gln Arg Arg Glu Gln Ala Asn Gly Glu Val ArgAla Cys Ser Leu Pro His Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys GlnAla Pro Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser His Trp Lys AsnCys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu Lvs Asn Ala Ser Asp Lys Arg Asn GlnGln Thr Ile Leu Gly Ser Pro Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly GlnGlnGly Ser Ile Glu Leu Glu Leu Gln Met Asn Arg Arg Tyr***
7.根據權利要求5所述的PPAR拮抗劑和激動劑的篩選方法，其特徵在於含人PPARγLBD和酵母轉錄因子GAL4-BD融合蛋白表達質粒pGBKT7-PPARγLBD具有如下序列(1)利用載體pGBKT7構建的pGBKT7-PPARγLBD質粒，具有如下核苷酸序列CGGTGCGGGCCTCTTCGCTATTACGCCAGATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCCTGAAAGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGgcggagatctccagtgatatcgaccagctgaatccagagtccgctgacctccgggccctggcaaaacatttgtatgactcatacataaagtccttcccgctgaccaaagcaaaggcgagggcgatcttgacaggaaagacaacagacaaatcaccattcgttatctatgacatgaattccttaatgatgggagaagataaaatcaaagttcaaacacatcacccccctgcaggagcagagcaaagaggtggccatccgcatctttcagggctgccagtttcgctccgtggaggctgtgcaggagatcacagagtatgccaaaagcattcctggttttgtaaatcttgacttgaacgaccaagtaactctcctcaaatatggagtccacgagatcatttacacaatgctggcctccttgatgaataaagatggggttctcatatccgagggccaaggcttcatgacaaagggagtttctaaagagcctgcgaaaagccttttggtgactttatggagcccaagtttgagtttgctgtgaagttcaatgcactggaattagatgacagcgacttggcaatatttattgctgtcattattctcagtggagaccgcccaaggtttgctgaatgtgaagcccattgaagacattcaagacaacctgctacaagccctggagctccagctgaagctgaaccaccctgagtcctcacagctgtttgccaagctgctccagaaaatgacagacctcagacagattgtcacggaacacgtgcagctactgcaggtgatcaagaagacggagacagacatgagtcttcacccgctcctgcaggagatctacaaggacttgtactagGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGTCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT(2)利用載體pGBKT7構建的pGBKT7-PPARγLBD質粒表達的胺基酸序列如下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 MetAla Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala AspLeu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lvs Ser Phe Pro Leu Thr Lys Ala LysAla Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn SerLeu Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys GluVal Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr GluTyr Ala Lys Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys TyrGly Val His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile SerGlu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp PheMet Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp LeuAla Ile Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile GluAsp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu SerSer Gln Leu Phe Ala Lys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His ValGln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gln Glu IleTyr Lys Asp Leu Tyr***
全文摘要
本發明涉及利用酵母雙雜交系統，通過測定α－半乳糖苷酶的活性大小篩選PPAR拮抗劑和激動劑的方法。該方法包括(1)CBP和酵母GAL4AD融合蛋白(pGADT7－CBP)的質粒構建；(2)PPARγLBD和酵母GAL4BD融合蛋白(pGBKT7－PPARγLBD)的質粒構建；(3)將質粒pGBKT7－PPARγLBD與質粒GADT7－CBP依次轉入酵母細胞AH109；(4)通過測定α－半乳糖苷酶的活性篩選化合物。
文檔編號C12Q1/34GK1629310SQ20031012270
公開日2005年6月22日 申請日期2003年12月19日 優先權日2003年12月19日
發明者沈旭, 蔣華良, 沈建華, 陳晴, 陳靜, 郭紅霞, 陳帥, 羅小民, 莊賢韓 申請人:中國科學院上海藥物研究所, 上海先導藥業有限公司