摘要
通过分析已知两种分枝杆菌的3-甾酮-Δ1-脱氢酶(kstD)碱基保守序列,设计简并引物,以甾醇降解菌株Mycobacterium neoaurumNwIB-01的DNA作模板,得到一个kstD基因片段,通过染色体走读,得到其完整的阅读框。以pUC18为异源表达载体,构建pTQ002表达质粒,在E.coliDH5α中对kstD进行了胞内活性表达,测定Mycobacteri-um neoaurumNwIB-01 kstD酶活为(0.37±0.05)U.mL-1,约为原酶活的一半,为构建高效转化生成3-酮基-1,4-二烯类固醇的基因工程菌奠定了基础。
The primers were designed through analyzing the genome sequence of two known 3-ketosteroid-Δ1-dehydrogenase in Mycobacterium,and the conservative region was obtained with Mycobacterium neoaurum NwIB-01 as the template.Through genome walking,the sequence after the conservative region about 1500 bp long was got.Then pTQ002 was constructed as the expression vector,and the kstD was expressed in E.coli DH5α.The 3-ketosteroid-Δ1-dehydrogenase activity of Mycobacterium neoaurum NwIB-01 was（0.37±0.05）U·mL-1,about half of the former one.
出处
《化学与生物工程》
CAS
2011年第7期39-43,共5页
Chemistry & Bioengineering
基金
中国博士后基金资助项目(20100470757)
