甲醇酵母PEG和电转化法

第24卷第3期河北大学学报(自然科学版)Vol.24 No.32004年5月Journal of Hebei University (Natural Science Edition)May 2004甲醇酵母PEG和电转化法李亚东,王会文,赵晓瑜(河北大学生命科学学院,河北保定07102)摘要:利用电击法和PEG法分别转化甲醇酵母，电击法的转化率达(DNA)400转化子/rg,明显高于PEG法,后者仅为0~20转化子/pg,而且操作更加快捷.关键词:甲醇酵母;转化;电穿孔;PEG中图分类号:Q 812文献标识码:A文章编号:1000- 1565(2004)03 - 0299-02甲醇酵母( Pichia pastoris )是集真核生物和原核生物表达系统的优点[41-3]于- -身的新- -代表达系统:它既具有原核生物的易于培养繁殖快、便于基因工程操作等优点,又具有真核生物翻译后的修饰加工机制,尤其可对表达的蛋白质进行简单的糖基化修饰.因此对于蚯蚓纤溶酶( Earthworm FibrinoLytie Enzymes, EFE)这-真核生物的糖蛋白而言,酵母表达系统无疑是较理想的表达体系.由于酵母有细胞壁,结构复杂,因此质粒向酵母的转化不同于大肠杆菌.经典的方法是通过制备原生质体进行转化[41.利用PEG(聚乙二醇)是近年来发展的- -种较为简便的方法,该法操作比较简单,但其转化率与PEG的相对分子质量大小有很大的关系,而且转化率较低.采用电击法,不仅操作更加快捷,而且转化率也大大提高.1材料与方法1.1 材料甲醇酵母GS115;pPIC9K( Iitrogen公司);LN- 101基因脉冲导入仪(天津理I学院).1.2方法1.2.1构建含有 EFE基因片段的p9K-EK7质粒根据EFE活性最佳组分的N-末端设计简并引物,以蚯蚓( Eisenia foetida )cDNA为模板,通过PCR获得该组分的编码DNA片段,并克隆到pPIC9K质粒中,构建成p9K - EK7质粒.1.2.2 电击法将p9K - EK7质粒转化到甲醇酵母接100 μ甲醇酵母GS115于10 mL YPD中,30 C培养过夜.第2天1%接种量转接至YPD中,30 C培养至菌液浓度达ODo0= 1.0左右.取1.5 mL菌液,室温5 kr/min离心1 min,用1 mL无菌冰水洗2次，再用1 mL(1 mol/L)}预冷的山梨醇洗-遍,最后用0.1 mL( 1 mol/L)预冷的山梨醇重新悬浮细胞.将20山(约5ug)经线性化的p9K- EK7质粒与80 uL细胞混匀,置于0.2 cm的电转杯中冰浴5 min. 在7 500 V/cm下,分别以5,10,20 pF电容进行电击转化,电击后迅速加入0.2 ml (1 mol/L)山梨醇,徐MD平板,30 C培养3~5 d.1.2.3 PEG/LiCl 法转化p9K- EK7质粒于甲醇酵母按1.2.2培养甲醇酵母GS115至菌液浓度达0D00= 1.0左右.取0.5 mL菌液，室温2 kr/min离心5min弃上清,用0.5mL无菌水重新悬浮菌体,室温2kr/min离心5min弃上清,用0.25mL(0.1mol/L)收稿日期:2003 -09 -01作者简介:李亚东(1977- ),男,河北滦平人,河北大学在读硕:士研究生.第3期蔡建国等:湿式催化氧化技术的研究进展●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(责任编辑:梁俊红)(上接第300页)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