甘油脱水酶再激活酶的克隆表达及活性鉴定  

Cloning and Expression of the Genes Encoding Glycerol Dehydratase Reactivase and Identification of its Biological Activity

作　　者：李雯君[1] 方柏山[1] 洪燕[1] 王晓霞[1] 林锦霞[1] 刘桂兰[1] 

机构地区：[1]工业生物技术福建省高等学校重点实验室(华侨大学),泉州362021

出　　处：《生物工程学报》2006年第6期950-955,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目(No.20276026;20446004);福建省科技计划项目重点项目基金(No.2003I020);华侨大学自然科学基金(No.03HZR2)资助。~~

摘　　要：运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a(+)上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。The gdrA, gdrB gene coding glycerol dehydratase reactivase factor were amplified by using the genomic DNA of KlebsieUa pneumoniae as the template. The gdrA and gdrB were inserted in pMD-18T to yield the recombinant cloning vector pMD-gdrAB. After the DNA sequence was determined, the gdrAB gene was subcloned into expression vector pET-28a（ ＋ ） to yield the recombinant expression vector pET-28gdrAB. Under screening pressure by ampicillin and kanamycin simultaneously, the activity of glycerol dehydratase reactivase was characterized by coexpression of pET-32gldABC, which carry the gldABC gene encoding glycerol dehydratase, and pET-28.gdrAB in E. coli BL21（DE3）.

关 键 词：甘油脱水酶 甘油脱水酶再激活酶 共表达 不相容双质粒 分子伴侣 

分 类 号：Q78[生物学—分子生物学]