酵母雙雜交系統(tǒng)技術(shù)介紹1課件

1、酵母雙雜交系統(tǒng)技術(shù)介紹1酵母雙雜交系統(tǒng)技術(shù)介紹 酵母雙雜交系統(tǒng)技術(shù)介紹1主要內(nèi)容酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: 關(guān)注蛋白質(zhì)酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: 為什么要研究蛋白互作？酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: clontech 酵母雙雜交系統(tǒng)的功能酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: clontech分子生物學(xué)工具clontechmatchmakermatchmaker酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: matchmaker gold 系統(tǒng)酵母雙雜交系統(tǒng)技術(shù)介紹1背景介紹: matchmaker gold系統(tǒng)原理reporter genereporter genereporter ge

2、ne酵母雙雜交系統(tǒng)技術(shù)介紹1主要內(nèi)容酵母雙雜交系統(tǒng)技術(shù)介紹1基本原理: 酵母雙雜交參考文獻(xiàn) nature. 1989 jul 20;340(6230):245-6. a novel genetic system to detect protein-protein interactions. fields s, song o. department of microbiology, state university of new york at stony brook, stony brook 11794.63000次引用酵母雙雜交系統(tǒng)技術(shù)介紹1基本原理: gal4轉(zhuǎn)錄因子的結(jié)構(gòu)adaddna-

3、bddna-bdgal4 gal4 蛋白具有兩個功能結(jié)構(gòu)域蛋白具有兩個功能結(jié)構(gòu)域bdbd結(jié)合結(jié)構(gòu)域結(jié)合結(jié)構(gòu)域adad激活結(jié)構(gòu)域激活結(jié)構(gòu)域酵母雙雜交系統(tǒng)技術(shù)介紹1基本原理: gal4轉(zhuǎn)錄因子的分子機理在在gal4 basedgal4 based雙雜交系統(tǒng)中雙雜交系統(tǒng)中: :正常情況在酵母細(xì)胞內(nèi)正常情況在酵母細(xì)胞內(nèi): :promotergal metabolic genegal uastranscriptionadbdreporter genegal4 ad酵母雙雜交系統(tǒng)技術(shù)介紹1基本原理: 三個啟動子，四個報告基因報告基因表達(dá)報告基因表達(dá)abaaba抗性篩選抗性篩選adeade營養(yǎng)篩選營養(yǎng)篩選h

4、ishis營養(yǎng)篩選營養(yǎng)篩選x-a-galx-a-gal藍(lán)白篩選藍(lán)白篩選m1g1g2酵母雙雜交系統(tǒng)技術(shù)介紹1主要內(nèi)容酵母雙雜交系統(tǒng)技術(shù)介紹1clontech mm系統(tǒng)的優(yōu)點smartsmart方法與同源重組同源重組技術(shù)結(jié)合構(gòu)建文庫，聰明的酵母自己做克隆。abaaba抗性篩選的添加，有效抑制背景生長，大大降低假陽性。文庫構(gòu)建于型酵母菌株型y187中，方便后續(xù)與a型菌株y2h gold進(jìn)行matingmating直接篩選。酵母雙雜交系統(tǒng)技術(shù)介紹1clontech mm系統(tǒng): 蛋白互作基本流程驗證蛋白互作驗證蛋白互作自己構(gòu)建文庫：自己構(gòu)建文庫：630490630490預(yù)制細(xì)菌文庫：預(yù)制細(xì)菌文庫：638

5、802638802（傳統(tǒng)型）（傳統(tǒng)型）預(yù)制酵母菌文庫：預(yù)制酵母菌文庫：630480630480（均一化，通用型）（均一化，通用型） clontechclontech蛋白互作相關(guān)試劑盒蛋白互作相關(guān)試劑盒文庫篩選文庫篩選文庫構(gòu)建文庫構(gòu)建酵母雙雜交系統(tǒng)：酵母雙雜交系統(tǒng)：630489630489酵母菌轉(zhuǎn)化試劑盒：酵母菌轉(zhuǎn)化試劑盒：630439630439酵母培養(yǎng)基（套裝）：酵母培養(yǎng)基（套裝）：630495630495插入片段檢測：插入片段檢測：630497630497酵母質(zhì)粒提?。航湍纲|(zhì)粒提取：630467630467體內(nèi)驗證：體內(nèi)驗證：630489630489630305630305體外體外co-i

6、pco-ip驗證：驗證：630458630458630459630459631723631723631604631604酵母雙雜交系統(tǒng)技術(shù)介紹1文庫構(gòu)建: clontech mm文庫構(gòu)建方法 smart smart合成合成cdnacdna共轉(zhuǎn)化，涂板共轉(zhuǎn)化，涂板 培養(yǎng)，收集菌液培養(yǎng)，收集菌液 文庫分裝，凍存文庫分裝，凍存1ml mating1ml matingaaaaaaaaaaaaaaaaaattttttttttttttttccccccaaaaaaaaaaaaaaaaaattttttttttttttttggggggccccccaaaaaaaaaaaaaaaaaattttttttttttttt

7、tggggggld-pcrld-pcrccccccaaaaaaaaaaaaaaaattttttttttttttttggg酵母雙雜交系統(tǒng)技術(shù)介紹1 smart smart合成合成cdnacdna共轉(zhuǎn)化，涂板共轉(zhuǎn)化，涂板 培養(yǎng)，收集菌液培養(yǎng)，收集菌液 文庫分裝，凍存文庫分裝，凍存1ml mating1ml mating自己自己做克隆做克隆sd/leu-sd/leu-y187y187同源重組同源重組smart cdna smart cdna 兩兩小小時時構(gòu)構(gòu)建建文文庫庫文庫構(gòu)建: clontech mm文庫構(gòu)建方法酵母雙雜交系統(tǒng)技術(shù)介紹1 smart smart合成合成cdnacdna共轉(zhuǎn)化，涂板共

8、轉(zhuǎn)化，涂板 培養(yǎng)，收集菌液培養(yǎng)，收集菌液 文庫分裝，凍存文庫分裝，凍存1ml mating1ml matingsd/leu-sd/leu-文庫構(gòu)建: clontech mm文庫構(gòu)建方法酵母雙雜交系統(tǒng)技術(shù)介紹1文庫構(gòu)建: clontech mm自制文庫篩選 smart smart合成合成cdnacdna共轉(zhuǎn)化，涂板共轉(zhuǎn)化，涂板 培養(yǎng)，收集菌液培養(yǎng)，收集菌液 文庫分裝，凍存文庫分裝，凍存1ml mating1ml matingy187y187，ad ad 文庫文庫y2y2h goldh gold，bdbd誘餌培養(yǎng)液誘餌培養(yǎng)液酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: clontech mm文庫篩選6304

9、89 630489 matchmaker matchmaker gold yeast two-hybrid systemgold yeast two-hybrid system630489630489y187y187y2h goldy2h gold三個載體三個載體陽性對照，陽性對照，陰性對照。陰性對照。bd vectorbd vector：pgbkt7pgbkt7ad vector: pgadt7ad vector: pgadt7630439630439極大提高轉(zhuǎn)化率極大提高轉(zhuǎn)化率可以單獨購買可以單獨購買酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: clontech mm 系統(tǒng)質(zhì)粒誘餌載體誘餌載體in

10、y2h goldin y2h gold酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: 誘餌載體構(gòu)建及其自激活檢測y2y2h goldh gold誘餌載體誘餌載體酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: mating方法篩選y187y187 型型ddoddo篩選篩選二倍體二倍體y2h goldy2h golda a 型型mating mating 接合反應(yīng)接合反應(yīng) 酵母的有性生殖過程，酵母的有性生殖過程，clontechclontech利用這個過程，實現(xiàn)了文庫篩選。利用這個過程，實現(xiàn)了文庫篩選。 ad ad bd bd mixmixmatingmatingqdoqdo篩選篩選酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: 均

11、一化通用文庫篩選均一化通用型文庫均一化通用型文庫高豐度與低豐度均衡高豐度與低豐度均衡大大降低工作量大大降低工作量大大降低假陽性大大降低假陽性更高的基因代表性更高的基因代表性normalizeduniversalpre. libraryy187y187，ad ad 文庫文庫y2y2h goldh gold，bdbd誘餌培養(yǎng)液誘餌培養(yǎng)液酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: 傳統(tǒng)細(xì)菌文庫篩選文庫培養(yǎng)文庫培養(yǎng)提取質(zhì)粒提取質(zhì)粒轉(zhuǎn)化酵母菌轉(zhuǎn)化酵母菌 取1ml菌液按照一定比例用lb稀釋，150ul/板進(jìn)行涂板，過夜培養(yǎng)。將提取的質(zhì)粒轉(zhuǎn)化y187，獲得酵母文庫。matingmating篩選篩選將文庫與誘餌培養(yǎng)液

12、混合，進(jìn)行mating。涂篩選板，挑取陽性克隆子。刮取平板上的菌落，提取質(zhì)粒。酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: 驗證陽性克隆子（pcr確認(rèn)）25ul 2mix30 pcr30 pcrcyclescycles25ul h2o630497 630497 matchmaker matchmaker insert check pcr mix2insert check pcr mix2酵母雙雜交系統(tǒng)技術(shù)介紹1文庫篩選: 酵母質(zhì)粒提取630467 630467 e easy yeast plasmid isolation kit asy yeast plasmid isolation kit 酵母雙雜交

13、系統(tǒng)技術(shù)介紹1驗證互作: 體內(nèi)驗證seapseap630305 630305 mammalian mammalian matchmaker two-matchmaker two-hybrid assay kit 2hybrid assay kit 2酵母雙雜交系統(tǒng)技術(shù)介紹1驗證互作: 體外co-ip驗證baitacgfp1acgfp1plplpreypreyprotein a/gagarose beadsmatchmaker chemiluminescent co-ip vector setmatchmaker chemiluminescent co-ip vector set（6304586

14、30458）matchmaker chemiluminescent co-ip kitmatchmaker chemiluminescent co-ip kit（630459630459）prolabel chemiluminescent detection kit prolabel chemiluminescent detection kit （631723631723） 酵母雙雜交系統(tǒng)技術(shù)介紹1驗證互作: 體外co-ip驗證實驗流程plpl檢測檢測酵母雙雜交系統(tǒng)技術(shù)介紹1驗證互作: fret分析632466 632466 pdsredpdsred-monomer-c1 vector-mon

15、omer-c1 vector632470 632470 pacgfp1pacgfp1-c1-vector-c1-vector酵母雙雜交系統(tǒng)技術(shù)介紹1clontech matchmakerclontech matchmaker系統(tǒng)產(chǎn)品選擇指南系統(tǒng)產(chǎn)品選擇指南酵母雙雜交系統(tǒng)技術(shù)介紹1the frigida complex activates transcription of flc, a strong flowering repressor in arabidopsis, by recruiting the frigida complex activates transcription of fl

16、c, a strong flowering repressor in arabidopsis, by recruiting chromatin modification factorschromatin modification factors k choi, j kim, hj hwang, s kim - the plant cell , 2011 - am soc plant biol extracellular signal-regulated kinase 8 (erk8) controls estrogen-related receptor extracellular signal

17、-regulated kinase 8 (erk8) controls estrogen-related receptor ( (errerr) ) cellular localization and inhibits cellular localization and inhibits its transcriptional activityits transcriptional activity m rossi, d colecchia, c iavarone, a strambi - journal of biological , 2011 asbmbwsb-1, a novel il-

18、21 receptor binding molecule, enhances the maturation of il-21 receptorwsb-1, a novel il-21 receptor binding molecule, enhances the maturation of il-21 receptor h nara, t onoda, m rahman, a araki - cellular , 2011 elsevierfhl1 interacts with oestrogen receptors and regulates breast cancer cell growt

19、hfhl1 interacts with oestrogen receptors and regulates breast cancer cell growth l ding, c niu, y zheng, z xiong - journal of cellular , 2011 - wiley online libraryrab4 interacts with the human pglycoprotein and modulates its surface expression in multidrug resistant k562rab4 interacts with the huma

20、n pglycoprotein and modulates its surface expression in multidrug resistant k562 cells cells c ferrndizhuertas - journal of cancer, 2011 - wiley online library6. duplicated c-class mads-box genes reveal distinct roles in gynostemium development in cymbidium ensifolium 6. duplicated c-class mads-box

21、genes reveal distinct roles in gynostemium development in cymbidium ensifolium (orchidaceae)(orchidaceae) sy wang, pf lee, yi lee, yy hsiao - plant and cell , 2011 - jpn soc plant physiolprotein interaction domain mapping of human kinetochore protein blinkin reveals a consensus motif for binding of

22、protein interaction domain mapping of human kinetochore protein blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins bub1 and bubr1spindle assembly checkpoint proteins bub1 and bubr1 t kiyomitsu, h murakami - molecular and cellular , 2011 - am soc microbiol clontech matchmakerclontech matchmaker系統(tǒng)近期參考文獻(xiàn)系統(tǒng)近期參考文獻(xiàn)酵母雙雜交系統(tǒng)技術(shù)介紹1主要內(nèi)容酵母雙雜交