透明颤菌血红蛋白基因(vgb)在阿维链霉菌的整合表达

Integrative expression of Vitrescilla hemoglobin gene in Streptomyces avermitilis

pHZ1276是透明颤菌血红蛋白基因(vgb)的诱导型表达载体,携带有硫链丝菌素诱导的强启动子PtipA和ФC31int,attP基因使载体整合在染色体上。本研究利用组成型启动子ermP1P2替代pHZ1276上的诱导型启动子PtipA构建了组成型表达血红蛋白基因的质粒pHZ1291。将vgb基因去掉后构建了组成型链霉菌表达载体pHZ1294。将pHZ1276,pHZ1291导入阿维链霉菌(Streptomycesavermitilis)NRRL8165,所得重组子经Southern杂交验证后,蛋白粗提物经Western杂交和CO结合实验表明vgb基因在该菌中表达出了有活性的VHb蛋白。

pHZ1276 is Vitrescilla globin gene (vgb) inducible expression vector of Streptomyces, which contains strong promoter PtipA induced by thiostrepon and two integrate-related gene: int, attP from phageФC31. In this research, pHZ1291, a plasmid with constitutive promoter ermP 1P 2for vgb gene expression was constructed by replacement of PtipA of pHZ1276. After deleting vgb gene from pHZ1291, constitutive expression vector of Streptomyces pHZ1294 was constructed. pHZ1276 and pHZ1291 were introduced into S.avermitilis NRRL8165 through conjugation. Recombinants were confirmed by Southern blotting, which demonstrated that this vgb gene has integrated into chromosome of S.avermitilis. Expression of vgb gene was demonstrated by Western blotting and carbon monoxide binding analysis. Western blotting and carbon monoxide binding analysis of the produced proteins of the recombinant strain showed that active VHb protein has been expressed in S.avermitilis.