嗜盐嗜碱芽孢杆菌NTT76启动子和信号肽序列的克隆及表达

嗜盐嗜碱芽孢杆菌NTT76启动子和信号肽序列的克隆及表达 Cloning,Expression of Promoter and Signal Peptide Sequence from Haloalkaliphilic Bacillus NTT76 陈建军 CHEN Jian-jun 1 2 first-author 曹军卫 CAO Jun-wei 1 2 苏莉 SU Li 1 2 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 利用启动子信号肽探测型质粒 p GPB14 ,从嗜盐嗜碱芽孢杆菌 NTT76的总基因组中克隆具有启动子和信号肽功能的 DNA片段 .共得到 4 1个转化子 ,其氨苄青霉素抗性水平在 10 0 0～ 12 0 0 0 m g/ L之间 .从 3个不同抗性水平的转化子中提取重组质粒 ,酶切显示 ,外源插入片段在 10 0～ 5 0 0 bp之间 .将重组质粒转化枯草芽孢杆菌 ,也同样使枯草芽孢杆菌具有分泌β-内酰胺酶的功能 .β-内酰胺酶活力测定表明 ,大肠杆菌产生的酶主要积累在周质空间 ,而枯草芽孢杆菌产生的酶主要分泌在胞外 .对外源插入片段进行序列测定发现 ,所有片段中均含有启动子区、核糖体结合位点和信号肽编码区域 . Promoter and signal peptide function fragments from Haloalkaliphilic Bacillus sp.NTT76 were cloned in Escherichia coli using a promoter-signal sequence probe vector pGPB14. Forty-one clones were obtained,whose level of resistence to Amp ranged from 800 1 2 000 g/L. The recombinant plasmids from three different Amp r level clones were isolated. Restriction analysis showed the size of inserted fragment ranged for 100 5 00 bp. The recombinant plasmids were transformed and showed the same secretion function in B.subtilis. The amount and localization of β-lactamase were determined in E.coli and B.subtilis using the β-lactamase distribution assay. The results showed that the β-lactamase activities of E.coli are mainly in periplasm while the enzyme produced by B.subtilis secrects extracellularly. The sequencing data of the three cloned fragments showed that all fragments contain promoters, ribosome binding sites and signal peptide function fragments. 嗜盐嗜碱芽孢杆菌 启动子 信号肽 haloalkaliphilic Bacillus promoter signal peptide Q785 2001-04-01 2021-04-01 4