β-葡萄糖苷酶在酿酒酵母表面上的展示及酶学性质的研究  

Displaying of β-Glucosidase on Yeast Cell Surface and Studies on Propeertics of Enzyme

作　　者：姚淑敏[1] 倪娜[1,2] 徐朝阳[1] 杨革[1] 

机构地区：[1]曲阜师范大学生命科学学院,山东省曲阜市273165 [2]南京林业大学江苏省制浆造纸科学与技术重点实验室,江苏省南京市310037

出　　处：《曲阜师范大学学报（自然科学版）》2011年第3期81-86,共6页Journal of Qufu Normal University(Natural Science)

基　　金：江苏省制浆造纸科学与技术重点实验室开放基金(200901);曲阜师范大学博士启动基金资助项目

摘　　要：根据裂殖酵母的β-葡萄糖苷酶的基因序列和酒精酵母菌表面展示质粒载体pYD1上的多克隆位点设计引物,从裂殖酵母中提取总RNA,然后反转录扩增得到β-葡萄糖苷酶基因(bglA),把该基因连接到表面展示质粒载体pYD1上,成功构建了重组质粒pYD1-bglA,并转化用于表面展示的酵母EBY100菌株中,得到酵母转化子,提取酵母菌的DNA通过PCR扩增证实了β-葡萄糖苷酶基因已整合到酵母基因组中.以不含载体和含空载体的酵母菌为对照,加2%(w/v)的半乳糖诱导48 h后,利用免疫荧光法在荧光显微镜下检测到了来源于裂殖酵母的β-葡萄糖苷酶展示在酵母菌细胞表面,并对其酶学性质进行分析研究.The primers were designed according to β-glucosidase gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1,which is a vector used for protein surface display on Saccharomyces cerevisiae cells.The gene encoding bglA was amplified by Rt-PCR using the genomic RNA of Schizosaccharomyces pombe.The PCR product was inserted into the plasmid pYD1.Then the construction was transformed into the yeast strain EBY100.After the target genes in the transformants were induced with 2% galactose for 36 hours,the display of the blgA protein on the yeast surface was confirmed by immnofluorescence with fluorescence microscope,yeast cells containing no vector and empty vector as controls,and the characteristics of the enzyme was analysed.

关 键 词：裂殖酵母 Β-葡萄糖苷酶基因 酵母菌表面展示系统 免疫荧光法 酶活测定 

分 类 号：Q78[生物学—分子生物学]