Taq DNA耐热聚合酶在大肠杆菌中的克隆和高表达  

Cloning and Expression of Taq DNA Polymerase Gene in Escherichia coli

作　　者：杜汉森[1] 季朝能[1] 徐迈[1] 马匆匆 郑佐华[1] 毛裕民[1] 

机构地区：[1]复旦大学遗传学研究所,上海200433

出　　处：《生物工程学报》1996年第4期503-507,共5页Chinese Journal of Biotechnology

摘　　要：从水生栖热菌(Thermus aquaticus)YT-1中分离得到的Taq DNA 聚合酶是一种广泛应用于PCR的耐热DNA聚合酶。由于天然菌株酶产量较低,培养条件要求严格,酶的纯化过程极为繁琐而使产品成本较高,因而促使人们构建适合于大规模生产的基因工程菌株。已有人分别通过不同的途径,使用不同的载体,成功地在大肠杆菌菌株中表达了Taq 耐热DNA聚合酶基因。我们通过与前人不同的途径,把这一基因克隆到大肠杆菌的载体质粒pJLA503上并使其得以高表达,为降低生产成本和进一步研究该酶的各种特性提供了有利条件。Using phagemid pUC118, a Genomic library of Thermus aquaticus strain YT-1 constructed, and according to the reported sequence of the Taq thermostable DNA polymerase gene,eight primers were designed and synthesized from which four pair were used for PCR reaction. Through three rounds of screening, a clone which contain the whole Taq gene was obtained. In addition,two mutagenic primer was syrthesized according to the nucleotide sequence of the 5'-end and 3'-end fo the gene and Nde I and Sal I cleavage sites were created by means of oligonucleotide-mediated mutagenesis. The Taq gene was then subcloned in-fram into the high expression vector pJLA503 making use of these cleavage sites. With the transcription and translation system of pJLA503, the expression of Taq DNA polymerase in E. coli can reach 4472 u/mg protein.

关 键 词：DNA聚合酶 耐热聚合酶 基因克隆 水生栖热菌 

分 类 号：Q555.03[生物学—生物化学]