节杆菌BT801基因文库构建及其乙内酰脲酶基因分离与表达  

Construction of Gene Library of Arthrobacter BT801 and Isolation & Expression of Hydantoinase Gene

作　　者：郝淑凤[1,2] 张惟材[1] 袁红杰[1] 王恒梁[1] 黄留玉[1] 

机构地区：[1]军事医学科学院生物工程研究所 [2]中国科学院沈阳应用生态研究所沈阳110016

出　　处：《生物工程学报》2003年第3期281-285,共5页Chinese Journal of Biotechnology

摘　　要：L 乙内酰脲酶产生菌节杆菌BT80 1的染色体DNA经Sau3AI部分酶切后分离 30kb左右的片段 ,与经HpaI和PstI酶切的黏粒载体pKC5 0 5进行连接 ,将连接产物用包装蛋白包装 ,转染大肠杆菌DH5α得到 10 0 0 0多个转化子 ,构建成节杆菌BT80 1的基因组文库。通过薄层层析等方法筛选得到了 1个阳性克隆 ,通过亚克隆得到了乙内酰脲酶的完整基因 。Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau 3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with λ phage package protein and transfecting E.coli DH5α. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.

关 键 词：乙内酰脲酶 节杆菌 基因文库 基因表达 

分 类 号：Q782[生物学—分子生物学]