一种β-葡萄糖苷酶基因在大肠杆菌和巴斯德毕赤酵母中的克隆与鉴定  

Cloning and identification of a kind of β-Glucosidase Gene in Escherichia coli and Pichia pastoris

作　　者：邵金辉[1] 赵云[2] 韩金祥[3] 柳明洙[1] 董志扬[2] 

机构地区：[1]中科院微生物研究所 [2]山东省医药生物技术研究中心国家卫生部生物技术药物重点实验室,济南250062 [3]延边大学医学院

出　　处：《南通大学学报（医学版）》2005年第1期37-39,共3页Journal of Nantong University(Medical sciences)

摘　　要：目的 :克隆扣囊复膜孢酵母的β-葡萄糖苷酶基因 (BGL 1) ,为β-葡萄糖苷酶基因的表达作准备。方法 :用 PCR方法从扣囊复膜孢酵母的总 DNA中扩增得到 β-葡萄糖苷酶基因连接到 p GEM- T载体上 ,用限制性内切酶切下目的基因 ,插入到巴斯德毕赤酵母表达载体 p PIC9K中 ,使之位于α-因子信号肽下游 ,且与之同框 ,构建成重组质粒 p SHL9K。再将 β-葡萄糖苷酶基因在大肠杆菌和巴斯德毕赤酵母中进行克隆 ,并进行鉴定。结果 :重组p PIC9K转化大肠杆菌和巴斯德毕赤酵母后 ,进行酶切和 PCR鉴定 ,证明形成了目的基因的克隆。结论 :应用大肠杆菌和巴斯德毕赤酵母作为受体菌 ,p PIC9K为载体 ,成功克隆了Objective:To clone the β-glucosidase gene (BGL1) of Sacchromycopsis fibuligera for the expression of the β-glucosidase gene.Methods:The β-glucosidase gene was amplified with PCR from the total DNA of Sacchromycopsis fibuligera, and was linked into pGME-T vector.After the target gene was cut down by restriction endonuclease from recombinant pGME-T vector, BGL1 was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K of Pichia pastoris.Thus,the recombinant plasmid pSHL9K was obtained. Then, the β-glucosidase gene was cloned in Escherichia coli and Pichia pastoris and was identified.Results:The recombinant plasmid pSHL9K was transformed into Escherichia coli and Pichia pastoris. Then , the cloned target gene was confirmed by cutting of restriction endonuclease and PCR analysis.Conclution:The β-glucosidase gene was cloned successfully by using pPIC9K as the vector and by using Escherichia coli and Pichia pastoris as host bateria.

关 键 词：Β-葡萄糖苷酶 基因 聚合酶链反应 克隆 载体 

分 类 号：Q785[生物学—分子生物学]