蓝藻热休克诱导高效表达系统的构建

Construction of High Level Expression System by Heat Shock Induced in Cyanobacterium

以聚球藻Synechococcussp .PCC794 2作为基因工程受体菌 ,构建了热休克诱导的高效表达系统。用PCR法扩增并克隆出Synechococcussp .PCC794 2自身的热休克基因 groESL操纵子的强启动区 (2 4 0bp) ,并紧靠其后组装上泛素 (UB)融合的胸腺素α1(Tα1)目的基因 ,在下游组装有rbcSpolyA终止子。此外 ,还组装了一个完整的卡那霉素抗性基因作为筛选标记基因。这样 ,就构建了完整的蓝藻的基因表达系统。经转化蓝藻Synechococcussp .PCC794 2细胞 ,在 4 2℃热诱导 30min后 ,目的基因UB Tα1得到较高水平表达 ,表达量约占可溶性蛋白的 8.7%

A high level expression system by heat shock induced had been constructed for Synechococcus sp. PCC7942. First, the strong promoter (240bp) of heat shock gene groESL operon from Synechococcus sp. PCC7942 was cloned by PCR. Then the ubiquitin fused thymosinα 1 gene(UB Tα 1)?rbcS polyA terminator were subcloned sequentially in the downstream of groESL promoter. The kanamycin resistance gene, which as the marker gene, was also subcloned into this expression system to obtain the expression vector pEUTMT 1. Induced by 42℃ heat shock for 30 min, the level of UB Tα 1 protein reached a value of 8.7% of the total soluble protein in the transformants of Synechococcus sp. PCC7942. The result of the experiment demonstrates that the heat shock induced high level expression system of Synechococcus sp. PCC7942 was constructed successfully.