摘要
将人尿激酶原基因连在PpsbA启动子之后 ,再将启动子连同基因克隆入整合载体pTZ18中。pTZ18_8中含有一段来源于集胞藻 6 80 3的psbB基因片段作为整合平台。将整合表达载体用直接转化的方法转入聚球藻Syne chococcussp .PCC 70 0 2中。经氨苄青霉素筛选并扩大培养后的转化子进行DNA斑点印迹及Western印迹 ,证明了基因的存在及表达。菌体破碎后的上清液有较高的溶解纤维蛋白的活性 ,说明表达产物未形成包含体。经ELISA测定 ,表达量为每克鲜藻 2× 10 -5～ 3× 10 -5g蛋白。
Pro_urokinase (pro_UK) gene was ligated with promoter PpsbA and cloned into the integrative vector pTZ18_8, which contained a psbB gene fragment from Synechocystis sp. PCC 6803 as the integrative platform. The expression vector was transferred into Synechococcus sp. PCC 7002 via natural transformation. Transformants conferring ampicillin resistance were amplified and then analyzed. DNA dot blot and Western blot demonstrated the existence and expression of pro_UK gene. The supernatant from crude cell extract showed thrombolytic activity, indicating that the expression product did not form inclusion bodies. According to the results of ELISA, expression of pro_UK was about 2×10 -5 -3×10 -5 g per gram of wet cells.
基金
国家高科技海洋863项目!( 819_0 2_0 4)&&
关键词
尿激酶
溶栓剂
聚球藻7002
克隆
原基因表达
pro_urokinase
thrombolytic agent
Synechococcus sp. PCC 7002
integrative vector
genetic engineering
