儿茶酚双加氧酶基因大肠杆菌表达体系研究  

STUDY ON EXPRESSION SYSTEM OF CATECHOL 2,3-DIOXYGENASE GENE IN E.coli

作　　者：熊爱生[1] 彭日荷[1] 耿其芳[1] 李贤[1] 范惠琴[1] 姚泉洪[1] 

机构地区：[1]上海市农业遗传育种重点实验室

出　　处：《上海农业学报》2003年第2期1-4,共4页Acta Agriculturae Shanghai

摘　　要：从恶臭假单孢菌 (Pseudomonassp .S 47)的DNA中扩增得到 92 4bp编码儿茶酚双加氧酶的基因片段 ,将其构建入 pBluescriptSK载体 BamHI和 SacI位点间。测序结果显示与Kim ( 2 0 0 0 )报道一致。将该基因插入带庆大霉素抗性基因启动子和t1t2终止子载体pG2 5 1、分别带ompA和EAP信号肽、lpp和Gmr 启动子载体 pYF3 95和 pYF5 2 5 4中 ,构建组成型表达载体pG2 5 1,分泌型表达载体 pYFX1,pYFX2。酶活测定结果显示pG2 5 1为 665mu/mL ,pYFX1,pYFX2分别为 1815mu/mL ,10 15mu/mL。SDS PAGE电泳显示表达蛋白产物的分子量约为 3 5kD。分泌型表达载体pYFX1表达量大于 pYFX2 ,组成型表达载体A 924bp fragment encoding catechol 2,3-dioxygenase gene was got from the Pseudomonas sp. S-47 DNA by polymerase chain reaction (PCR) method. The PCR product was digested with BamHI and SacI, and inserted into the pBluescript SK vector. The sequencing result indicated that the nucleotide sequence of the xylE gene was the same as that reported by Kim in 2000. The gene was inserted into a vector pG251 with gentamicin resistance gene promoter and t1t2 terminator, and into vectors pYF395 and pYF5254 respectively with ompA and EAP signal sequences, and lpp and Gm r promoters, and so the constitutive expression vector pG251 and the secretion vectors pYFX1 and pYFX2 were constructed. The determination of enzyme activity showed that pG251 was 665 mu/mL, pYFX1 1815 mu/mL and pYFX2 1015mu/mL. The SDS-PAGE revealed that the expression product of xylE gene was about 35 kD protein.

关 键 词：儿茶酚双加氧酶基因 大肠杆菌 表达体系 基因克隆 基因表达 酶活分析 环境污染 

分 类 号：X512[环境科学与工程—环境工程] Q946.823[生物学—植物学]