大肠杆菌腺苷脱氨酶基因的克隆、表达与缺失

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MC4100　DNA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2kb DNA分子，同时转化JM83、MC4100、BL21（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、pUC18／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pUC18／JM83强的腺苷脱氨酶活性。

Cloning, Expression and Deletion of Ademosine Deaminase Gene in Escherica coli

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.