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 pHJLDH. The construct was introduced into B. bifidum by electroporation. Distinct band corresponding to the theoretical value of LDH was detected on SDSPAGE, and was further identified by Westernblotting. 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.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
2002年第6期660-663,共4页
Journal of Fudan University：Natural Science
