外源乳酸脱氢酶基因在双歧杆菌中的表达  

Expression of Heterologous ldh Gene in Bifidobacterium

作　　者：李宁军[1] 李琳[1] 江培 李盛翃 黄伟达[1] 

机构地区：[1]复旦大学生命科学学院生物化学系,上海200433

出　　处：《复旦学报（自然科学版）》2002年第6期660-663,共4页Journal of Fudan University：Natural Science

摘　　要：以大肠杆菌 双叉双歧杆菌的穿梭质粒pHJ为基础,插入从Lactococcuslactis总DNA中用PCR方法扩增得到的报告基因乳酸脱氢酶基因(ldh),构建了双叉双歧杆菌的表达质粒pHJ LDH,并采用电转化法导入双叉双歧杆菌进行了表达研究.对双叉双歧杆菌总蛋白的SDS PAGE分析结果表明,ldh报告基因的表达产物形成了明显的条带,Western blotting结果确认了该条带为ldh基因的产物.LDH的酶活性定量测定结果表明,重组双叉双歧杆菌的蛋白粗抽提液中LDH的比活性为5.5U/mg,菌中LDH的表达量约为2mg/L.此工作为今后双叉双歧杆菌的基因工程奠定了基础.To construct an expression vector for Bifidobacterium bifidum, lactate dehydrogenase (ldh) gene was cloned from Lactococcus lactis strain by polymerase chain reaction (PCR) as a report gene, and was cloned into a shuttle vector pHJ to generate the expression construct pHJLDH. The construct was introduced into B. bifidum by electroporation. Distinct band corresponding to the theoretical value of LDH was detected on SDSPAGE, and was further identified by Westernblotting. The specific activity of LDH was 5.5 U/mg in the crude extract of engineered bacteria and was estimated that about 2 mg of LDH was expressed in one liter culture medium. The results provided the possibility for expression of various heterologous genes in B. bifidum.

关 键 词：乳酸脱氢酶 双叉双歧杆菌 表达 基因 蛋白 

分 类 号：Q51[生物学—生物化学] Q786