【摘要】 将大肠杆菌的复制子rep和多克隆位点克隆到枯草杆菌质粒pGDV1的骨架上,即得到大肠杆菌枯草杆菌穿梭质粒载体pGDVM。在pGDVM上进行载体表达元件的构建,先后将P59启动子、核糖体结合位点SD和终止子克隆到pGDVM上得到穿梭表达载体GJ01。以β-半乳糖苷酶基因(bga)作为报告基因检测载体的表达活性,在大肠杆菌和枯草杆菌中β-半乳糖苷酶(Bga)酶活性最高达到75.3和83.2个密勒单位,表明所构建的表达载体具有较强的表达能力。以核糖体结合位点(SD2、SD3、SD4和SD5)代替表达载体GJ01-bga中的SD,对载体进行改造。所构建的GJ02-bga在大肠杆菌中的最大酶活性为253.8个密勒单位,GJ05-bga在枯草杆菌中的最大酶活性为135.4个密勒单位,表明所构建的载体具有较强的表达活性。由此可以得出不同的SD序列及其与起始密码子的距离不同程度地影响mRNA的翻译效率。更多还原

【Abstract】 Replicator of E.coli and MCS were cloned to pGDV1,a plasmid of Bacillus subtilis,to get a shuttle vector pGDVM.Used pGDVM as framework,P59 promoter,ribosome binding site(SD) and terminator were cloned to pGDVM,and then got the shuttle expression vector GJ01.Used bga as report gene to detect GJ01,the highest activity of Bga in E.coli and B.subtilis were partly 75.3 and 83.2 miller,indicated it had high express ability.We reconstructed the express vector through replace its SD by SD2,SD3,SD4 and SD5,the result was that: the activity of Bga of GJ01-bga in E.coli was 253.8 miller and the activity of Bga of GJ05-bga in B.subtilis was 135.4 miller.Then we could get that the distance between Shine-Dalgarno and start codon had affect differently the transformation of mRNA.更多还原