第24卷第3期河北大學(xué)學(xué)報(bào)(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(zhuǎn)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07102)摘要:利用電擊法和PEG法分別轉(zhuǎn)化甲醇酵母，電擊法的轉(zhuǎn)化率達(dá)(DNA)400轉(zhuǎn)化子/rg,明顯高于PEG法,后者僅為0~20轉(zhuǎn)化子/pg,而且操作更加快捷.關(guān)鍵詞:甲醇酵母;轉(zhuǎn)化;電穿孔;PEG中圖分類號(hào):Q 812文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):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(dá)系統(tǒng)的優(yōu)點(diǎn)[41-3]于- -身的新- -代表達(dá)系統(tǒng):它既具有原核生物的易于培養(yǎng)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(jī)制,尤其可對(duì)表達(dá)的蛋白質(zhì)進(jìn)行簡(jiǎn)單的糖基化修飾.因此對(duì)于蚯蚓纖溶酶( Earthworm FibrinoLytie Enzymes, EFE)這-真核生物的糖蛋白而言,酵母表達(dá)系統(tǒng)無(wú)疑是較理想的表達(dá)體系.由于酵母有細(xì)胞壁,結(jié)構(gòu)復(fù)雜,因此質(zhì)粒向酵母的轉(zhuǎn)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(zhuǎn)化[41.利用PEG(聚乙二醇)是近年來(lái)發(fā)展的- -種較為簡(jiǎn)便的方法,該法操作比較簡(jiǎn)單,但其轉(zhuǎn)化率與PEG的相對(duì)分子質(zhì)量大小有很大的關(guān)系,而且轉(zhuǎn)化率較低.采用電擊法,不僅操作更加快捷,而且轉(zhuǎn)化率也大大提高.1材料與方法1.1 材料甲醇酵母GS115;pPIC9K( Iitrogen公司);LN- 101基因脈沖導(dǎo)入儀(天津理I學(xué)院).1.2方法1.2.1構(gòu)建含有 EFE基因片段的p9K-EK7質(zhì)粒根據(jù)EFE活性最佳組分的N-末端設(shè)計(jì)簡(jiǎn)并引物,以蚯蚓( Eisenia foetida )cDNA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(gòu)建成p9K - EK7質(zhì)粒.1.2.2 電擊法將p9K - EK7質(zhì)粒轉(zhuǎn)化到甲醇酵母接100 μ甲醇酵母GS115于10 mL YPD中,30 C培養(yǎng)過(guò)夜.第2天1%接種量轉(zhuǎn)接至YPD中,30 C培養(yǎng)至菌液濃度達(dá)ODo0= 1.0左右.取1.5 mL菌液,室溫5 kr/min離心1 min,用1 mL無(wú)菌冰水洗2次，再用1 mL(1 mol/L)}預(yù)冷的山梨醇洗-遍,最后用0.1 mL( 1 mol/L)預(yù)冷的山梨醇重新懸浮細(xì)胞.將20山(約5ug)經(jīng)線性化的p9K- EK7質(zhì)粒與80 uL細(xì)胞混勻,置于0.2 cm的電轉(zhuǎn)杯中冰浴5 min. 在7 500 V/cm下,分別以5,10,20 pF電容進(jìn)行電擊轉(zhuǎn)化,電擊后迅速加入0.2 ml (1 mol/L)山梨醇,徐MD平板,30 C培養(yǎng)3~5 d.1.2.3 PEG/LiCl 法轉(zhuǎn)化p9K- EK7質(zhì)粒于甲醇酵母按1.2.2培養(yǎng)甲醇酵母GS115至菌液濃度達(dá)0D00= 1.0左右.取0.5 mL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離心5min棄上清,用0.25mL(0.1mol/L)收稿日期:2003 -09 -01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.第3期蔡建國(guó)等:濕式催化氧化技術(shù)的研究進(jìn)展●331peper 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 efluents in a trickle - bed reactor overa Ru/TiO2 catalyst[J]. Applied Catalyssi B: Enironmental,2001 ,31 :275 - 290.[29] DAEWON P,WONSEOK C. Decoloring dye wastewater with low temperature catalytic oxidation[J]. Wat Sci Tech, 199,40(4/5):115- 121.[30] LEI L,HU X,CHU H P,et al. Catalytic wet oxidation of dyeing and printing wastewater[]. Wat Sci Tech, 1997, 35(4):311-319.[31] FAJERWERG K,FOUSSARD J N,PERRARD A,et al. Wet oxidation of phenol by hydrogen peroxide:the key role of pH on .the catalytic behaviour of Fe- ZSM - 5[J]. Wat Sci Tech, 1997 ,35(4):103- 110.[32] TUNG LI HUANG,JORDAN M MACLNNES,KEITH R CLIFFE,et al. Nitrogen removal from wastewater by a catalytic ox-idation method[J]. Wat Res, 2001 ,35(9):2113- 2120.[33] 0XANA P PESTUNOVA,GALINA L ELIZAROVA,ZINFER R ISMAILOV ,et al. Detorication of water cntaining1,1-dimethyIhydrazine by ctalyic oxidation with dioxygen and hydrogen peroxide over Cu- and Fe- ontaing calyts[J]. Catal.ysis Today ,2002,75:219 - 225.Progress of Wet Catalytic Air Oxidation TechnologyCAI Jian-guo,LI Ai-min,ZHANG Quan-xing(State Key Laboratory of Pllution Control and Resource Reuse, School of Environment,Nanjing University , Nanjing 210093,China)Abstract:Wet catalytic air oxidation is a useful kind of advanced oxidation process for high srength, toxic,hazardous and non- biodegradable contaminants under milder conditions, which is developed on the basic of wetair oxidation. The basic concepts,mechanism and kinetis, cssicationn and aplication to wastewater treatmentof wet catalytic air oxidation are reviewed.Key words:wet catalytic air oxidation; wet air oxidation;catalyst; wastewater treatment(責(zé)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-yu(Colle of Life Sciences ,Hebei University , Baoding 071002 ,China)Astrat:letricporation and PEG transformation were used to transform methylotrophic yest rspetively .The ransforming rate(DNA) of etroporation method was 400 tasomants/ug, while the PEG methodwas only 0~ 20 transformants /pg.Key words:methylotrophic yeast; transformation; eletroporation; PEG