HBV PreS1-EGFP融合表达质粒高效转化毕赤氏酵母的研究  

Study on Efficient Transformation of Fusion Expression Plasmid of HBV PreS1-EGFP into Pichia Pastoris

作　　者：周小玲[1] 李子龙 孙平楠[1] 

机构地区：[1]汕头大学医学院,广东汕头515041 [2]中科院北京微生物研究所,北京100101

出　　处：《现代生物医学进展》2009年第18期3427-3428,F0003,共3页Progress in Modern Biomedicine

基　　金：国家自然科学基金(30801240);国家自然科学基金(30901609);广东省自然科学基金(No8451503102001823)

摘　　要：目的:实现HBV PreS1-EGFP融合表达质粒高效转化毕赤氏酵母。方法:分别将乙肝病毒前S1蛋白和绿色荧光蛋白克隆到表达载体pPIC9K,获得重组质粒pPIC9K-EGFP-preS1,SacI位点酶切线性化,采用醋酸锂(LiAc)和二硫苏糖醇(DDT)对巴斯德毕赤氏酵母Pichia Pastoris GS115预处理,在电压1.5kv、电容25F条件下进行电击转化。电击后立即迅速稀释在冰预冷的1M的山梨醇中,混匀后取100l的菌液均匀涂布YNBG平板,利用HIS4标记筛选转化子。结果:发现改进后的转化方法pPIC9K-EGFP-preS1的转化率提高到164×104个转化子/1μgpPIC9K-EGFP-preS1 DNA,是传统方法的6.6倍。结论:采用改进后的方法能有效的提高质粒pPIC9K-EGFP-preS1的转化率,为筛选高表达HBVPreS1-EGFP融合蛋白的转化子打下基础。Objective： To realize efficient transformation of fusion expression plasmid of HBV PreSI-EGFP into P. Pastoris. Methods： HBV PreSlgene and green fluorescent protein gene were inserted into the expression vector of pPIC9K to yield pPIC9K-EGFP-preS1. This recombinant plasmid was linearized by the enzyme of Sac I and transformed into P.Pastoris GS115 （pretreated with [,iAc and DDT） by electroporation at 1.5 kv and 25 F. After electroporation the cells were immediately diluted in the ice-cold sorbitol, spreaded onto the YNBG plate, and selected by HIS4 selection marker. Results： The improved method in which P. pastoris GS115 was pretreated with LiAc and DDT increased the transformation efficiency to 164×10^4 transformants/1 μg pP1C9K-EGFP-preS1 DNA, 6.6 times than the traditional method. Conlusions： The improved method can effectively elevate the transformation efficiency of pPICgK-EGFP-preS1 so as to provide the base for selecting transformants of high-level expression ofHBV PreSl-EGFP.

关 键 词：乙肝病毒前S1蛋白 高效转化 毕赤氏酵母 

分 类 号：Q78[生物学—分子生物学] R318.04[医药卫生—生物医学工程]