甲醇酵母PEG和電轉化法

第24卷第3期河北大學(xué)學(xué)報(自然科學(xué)版Vol 24 No. 32004年5月Journal of Hebei University(Natural Science Edition)May 2004甲醇酵母PEG和電轉化法李亞?wèn)|,王會(huì )文,趙曉瑜(河北大學(xué)生命科學(xué)學(xué)院河北保定071002摘要:利用電擊法和PEG法分別轉化甲醇酵母,電擊法的轉化率達(DNA)400轉化子/四,明顯高于PEG法,后者僅為0~20轉化子/四g,而且操作更加快捷關(guān)鍵詞:甲醇酵母;轉化;電穿孔;PEG中圖分類(lèi)號:Q812文獻標識碼:A文章編號:1000-1565(2004)03-0299-02甲醇酵母( Pichia pastoris)是集真核生物和原核生物表達系統的優(yōu)點(diǎn)-3于一身的新一代表達系統:它既具有原核生物的易于培養繁殖快、便于基因工程操作等優(yōu)點(diǎn),又具有真核生物翻譯后的修飾加工機制,尤其可對表達的蛋白質(zhì)進(jìn)行簡(jiǎn)單的糖基化修飾因此對于蚯蚓纖溶酶( Earthworm FibrinoLytic Enzymes,EFE)這一真核生物的糖蛋白而言酵母表達系統無(wú)疑是較理想的表達體系由于酵母有細胞壁結構復雜,因此質(zhì)粒向酵母的轉化不同于大腸桿菌經(jīng)典的方法是通過(guò)制備原生質(zhì)體進(jìn)行轉化4,利用PEG(聚乙二醇)是近年來(lái)發(fā)展的一種較為簡(jiǎn)便的方法,該法操作比較簡(jiǎn)單,但其轉化率與PEG的相對分子質(zhì)量大小有很大的關(guān)系,而且轉化率較低采用電擊法,不僅操作更加快捷,而且轉化率也大大提高1材料與方法1.1材料甲醇酵母GS15;pPIC9K( Invitrogen公司);LN-101基因脈沖導入儀(天津理工學(xué)院1.2方法1.2.1構建含有EFE基因片段的p9K-EK7質(zhì)粒根據EFE活性最佳組分的N-末端設計簡(jiǎn)并引物,以蚯蚓( Eisenia foetida)dNA為模板,通過(guò)PCR獲得該組分的編碼DNA片段并克隆到 pPIC9K質(zhì)粒中構建成p9K-EK7質(zhì)粒1.22電擊法將p9K-EK7質(zhì)粒轉化到甲醇酵母接100uL甲醇酵母GS15于10 mL YPD中,30℃培養過(guò)夜第2天1%接種量轉接至YPD中,30℃培養至菌液濃度達OD0=1.0左右取1.5mL菌液,室溫5kr/min離心1min,用1mL無(wú)菌冰水洗2次,再用1mL(1mOl1L)預冷的山梨醇洗一遍最后用01mL(1mo/)預冷的山剩醇重新懸浮細胞將20μL(約5pg)經(jīng)線(xiàn)性化的p9K-EK7質(zhì)粒與80μL細胞混勻,置于0.2cm的電轉杯中冰浴5min.在7500V/cm下,分別以5,10,20pF電容進(jìn)行電擊轉化電擊后迅速加入0.2mL(1moL)山梨醇涂MD平板,30℃培養3~5d1.2.3PEG/Cl法轉化p9K-EK7質(zhì)粒于甲醇酵母按12.2培養甲醇酵母GS115至菌液濃度達OD0=1.0左右取0.5mL菌液,室溫2 kr/min離心5min棄上清用0.5mL無(wú)菌水重新懸浮菌體,室溫2kr/min離上:田05mL(0.1molL)中國煤化工收稿日期:2003-09-01CNMHG作者簡(jiǎn)介:李亞?wèn)|(1977-),男,河北灤平人,河北大學(xué)在讀碩士研究生300河北大學(xué)學(xué)報(自然科學(xué)版)2004年LC洗2遍用轉化液(24pL50%PFG6003.6μ L I mol/L LiCl;2.5μL牛胸腺DNA;6線(xiàn)性質(zhì)粒,加水定容至36μL)懸浮菌體并混勻,30℃保溫30min,42℃再保溫25min,室溫2kr/min離心5min,棄上清.用0.1mL無(wú)菌冰水懸浮菌體,混勻后涂MD平板,30℃培養3~5d2結果與討論2.1電轉化法轉化甲醇酵母電轉化法是利用髙壓電脈沖使細胞膜發(fā)生瞬間的可逆穿孔,從而使游離的外源基因穿過(guò)細胞膜而轉移到細胞中去電穿孔操作對受體細胞產(chǎn)生的干擾很小,從而保持了活細胞的正常生理條件這是一種很有前途的基因轉化方法,目前已廣泛應用于細胞工程和基因工程方面本實(shí)驗中使用LN-101基因脈沖導入儀,采用7500V/m的電壓,0.2cm間距的轉化杯對甲醇酵母GS15進(jìn)行電擊轉化,發(fā)現在所采用的3個(gè)電容(5,10,20pF)中,電容為10m時(shí)的轉化率最高,轉化率(DNA)約為400個(gè)轉化子/p電擊法轉化結果如圖1所示轉化組對照組圖1電擊法轉化后MD平板生長(cháng)情況Fig 1 Efficiency of electric transformation for GS115實(shí)驗中使用的電擊轉化儀為天津理工學(xué)院生產(chǎn),與功能類(lèi)似的國外產(chǎn)品相比,具有價(jià)格便宜的優(yōu)點(diǎn),且轉化后出現的陽(yáng)性克隆的數量完全可以滿(mǎn)足下一步實(shí)驗的需要22PEG法轉化甲醇酵母雖然電擊轉化方法具有轉化效率高、簡(jiǎn)便等優(yōu)點(diǎn)但畢竟還需要特殊的電轉化設備本實(shí)驗中也采用PEG/LiCI法轉化,并獲得成功值得注意的是在該法中PEG是個(gè)關(guān)鍵的因素:除了濃度以外,PEG相對分子質(zhì)量的大小對轉化有很大影響,直接關(guān)系到轉化能否成功本實(shí)驗曾先后使用PEG1500,PEc4000和PEG6000進(jìn)行轉化,但只有PEG6000得到了轉化子與電擊轉化法對照該法的最大缺點(diǎn)在于轉化效率較低轉化率(DBA)一般在0~20轉化子g,僅為擊法的1%-5%原因主要在于甲醇酵母細胞存在較厚的細胞壁僅用PEGC誘導使外源DNA穿過(guò)厚厚的細胞壁進(jìn)入細胞相對比較難因此對于甲醇酵母表達系統來(lái)說(shuō),利用電擊轉化比較合適[]王會(huì )文趙曉瑜甲醇酵母基因表達系統[生命的化學(xué),2000,20(6):271-272[2]戴秀玉.一種值得注意的基因表達系統一巴斯德畢赤氏酵母[J[3]彭毅,步威康良儀甲醇酵母表達系統[生物技術(shù)通報,20中國煤化工4]盧圣棟現代分子生物學(xué)實(shí)驗技術(shù)[M].北京:高等教育出版社,19CNMHG(下轉第331頁(yè))第3期蔡建國等:濕式催化氧化技術(shù)的研究進(jìn)展33Ipaper and pulp industrial waste liquor[J] Applied Catalysis A: General, 2002, 236: 255-262[28] PINTAR A, BESSON M, GALLEZOT P Catalytic wet air oxidation of kraft bleach plant effluents in a trickle-bed reactor overa Ru/TiO catalyst[ J] Applied Catalyssi B: Environmental, 2001,31:275-290[29]DAEWON P, WONSEOK C Decoloring dye wastewater with low temperature catalytic oxidation[ J]. 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Detoxication of water containing 1, 1dimethylhydrazine by catalytic oxidation with dioxygen and hydrogen peroxide over Cu-and Fe-containing catalysts [J].Catalysis Today,2002,75:219-225Progress of Wet Catalytic Air Oxidation TechnologyCAl Jian-guo, LI Ai-min, ZHANG Quan-xingState Key Laboratory of Pollution Control and Resource Reuse, School of EnvironmentNanjing University, Nanjing 210093, China)Abstract: Wet catalytic air oxidation is a useful kind of advanced oxidation process for high strength, toxichazardous and non-biodegradable contaminants under milder conditions, which is developed on the basic of wetair oxidation. The basic concepts, mechanism and kinetics, classification and application to wastewater treatmentof wet catalytic air oxidation are reviewedKey words: wet catalytic air oxidation; wet air oxidation; catalyst; wastewater treatment(貴任編輯:梁俊紅)上接第300頁(yè))Electroporation and PEG Methods of Transformationfor Methlyotrophic YeastLI Ya-dong, WANG Hui-wen, ZHAo Xiao-yuCollege of Life Sciences, Hebei University, Baoding 071002, China)Abstract: Electricporation and PEG transformation were used to transform methylotrophic yeast respectivelyThe transforming rate(DNA)of electroporation method was 400 transformants/ug, while the PEG methodwas only 0-20 transformants /ugKey words: methylotrophic yeast; transformation; eletroporation: PEC中國煤化產(chǎn)任編輯:梁俊紅)CNMHG