嘌呤核苷磷酸化酶基因的克隆及原核表达载体的构建  

Cloning and expression of purine nucleoside phosphorylase in Escherichia coli

作　　者：李晓晖[1] 孙嘉康[1] 高彤[1] 任大明[1] 陈晓丽[2] 刘岩[2] 乔宾福[2] 

机构地区：[1]复旦大学遗传所遗传工程国家重点实验室,上海200433 [2]珍奥集团股份有限公司生物工程研究所,大连116620

出　　处：《工业微生物》2006年第3期27-31,共5页Industrial Microbiology

摘　　要：通过PCR方法从产气肠杆菌、胡萝卜软腐欧文氏菌、大肠杆菌扩增嘌呤核苷磷酸化酶(PNPase)基因,然后将扩增的约720bp的基因片段克隆到pET-28b表达载体上,构建重组PNPase的表达载体。核苷酸及推导的氨基酸序列分析表明,该基因在三个菌株之间有很高的同源性。SDS-PAGE电泳结果显示出明显的特异性蛋白质条带,其分子量约为29.8kDa.该载体的构建为进一步研究核苷及其类似物的生物合成奠定基础。The purine nucleoside phosphorylase（ EC 2.4.2.1 PNPase）gene of Enterobacter aerogenes ,Erwinia carotovora and E. coli was amplified by polymerase chain reaction（PCR）. The amplified fragment with about 720bp was cloned into the prokaryotic expression vector pET-28b（ ＋ ）. The three recombinant expression vectors that contained the target fragment were identified by digestion using restriction endonucleases and named as pP-8, pLErO-2 and pD1-1. The nucleotides and deduced amino acid sequences of the cloned PNPase gene from three strains were analyzed. The results showed that the gene had a relatively high homology with the corresponding gene from Enterobacteriaceae. The construction of recombinant PNPase will provide a basis for the research on biocatalysed synthesis of nucleosides and modified nucleosides.

关 键 词：嘌呤核苷磷酸化酶 基因克隆 表达载体构建 

分 类 号：Q78[生物学—分子生物学]