文献类型：期刊文献

中文题名：大肠杆菌腺苷脱氨酶基因的克隆、表达与缺失

英文题名：Cloning, Expression and Deletion of Ademosine Deaminase Gene in Escherica coli

作者：钱红亮[1];曾洋[1];叶江[1];张惠展[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室 分子克隆实验室,上海200237

年份：2002

卷号：42

期号：6

起止页码：686

中文期刊名：微生物学报

外文期刊名：Acta Microbiologica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；PubMed;

语种：中文

中文关键词：腺苷脱氨酶;ATP;基因缺失;代谢途径;基因工程;克隆;表达;途径工程

外文关键词：Adenosine deaminase, ATP, Gene deletion, Pathway engineering

摘要：利用途径工程的基本原理 ,拟在大肠杆菌核苷酸代谢途径中构建腺苷 (AR)转化为腺苷三磷酸 (ATP)的新途径 ,故需使细胞内的腺苷脱氨酶基因 (add)缺失。通过构建大肠杆菌MC41 0 0DNA的基因文库 ,筛选得到含腺苷脱氨酶基因的DNA片段。构建表达质粒pBD1和pBD2并实现了表达。在此基础上构建了add基因缺失的带卡那霉素抗性基因的线性 5 2kbDNA分子 ,同时转化JM83、MC41 0 0、BL2 1 (DE3 )。经遗传稳定性实验和DNA分子杂交鉴定 ,确认得到了来自JM83的两株add基因缺陷株J1和J2。再对菌株J1、pUC1 8/JM83、pBD1 /JM83的细胞粗提液做腺苷脱氨酶的酶活鉴定比较 ,结果表明则没有腺苷脱氨酶活性 ,pBD1 /JM83有比pUC1
Based on the principles of metabolic engineering, a new pathway of transforming adenosine to ATP is attempted to be built in the nucleotides metabolism of %E.coli %. Deletion of adenosine deaminase gene ( %add %) is to be needed. The %add % gene from %E.coli % MC4100 was cloned by constructing gene library and expressed through constructing two plasmids pBD1 adn pBD2. In order to get the %add %-defective strain, a 5 ^2kb DNA sequence including fragmental %add % gene and marker gene %Km +r % was made and transferred into JM83, MC4100 and BL21(DE3). The genetic stability and DNA hybridization experiment proved that two %add %-defective strain J1 and J2 come from JM83 were found. The enzyme assays indicated that strain J1 lost the adenosine deaminase activity and pBD1/JM83 showed higher adenosine deaminase activity than pUC18/JM83.