低分子量尿激酶与Annexin V的融合基因在家蚕杆状病毒表达系统中的表达

Expression of Prourokinase (144-411)-Annexin V Chimeras in Bombyx mori Baculovirus Expression Vector System

将低分子量尿激酶与膜联蛋白 (AnnexinV)的融合基因重组到家蚕杆状病毒转移载体pBacPAK8中 ,获得重组转移载体pBacPAK UKAV ,并与线性化的病毒Bm PAK6DNA共转染家蚕细胞 ,获得重组病毒BacPAK UKAV。将重组病毒BacPAK UKAV感染家蚕培养细胞 (MOI=10 )和 5龄幼虫 (10 5pfu/头 ) ,Western印迹方法表明表达产物大小约为 6 9kD。用纤维蛋白平板溶圈法测定表达产物的纤溶活性 ,其融合蛋白具有明显的纤溶活性 ,约为 5× 10 -5U/个细胞。用APTT法测定表达产物的抗凝活性 ,比野生病毒Bm PAK6感染表达产物的APTT时间延长了 1倍以上 ,表现出明显的抗凝活性。体外实验表明 ,家蚕培养细胞和幼虫表达的重组低分子量尿激酶与AnnexinV融合蛋白具有溶栓与抗栓双功能。研究结果为今后进一步探索具有溶栓抗栓功能的血栓药物提供了新的方向。

Prourokinase (144 411) Annexin V Chimeras fuse gene was inserted into Bombyx mori baculovirus transfer vector pBacPAK8 and co transfected with lineared DNA of Bm BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus BacPAK UKAV was obtained. It was identified by Southern Blotting. The BmN cell (MOI=10) and silkworm larvae at the fifth instar (10 5 pfu/larva) were infected by the recombinant virus BacPAK UKAV. The biological activity of the protein product was determined by APTT and fibrin well assays. The results showed the inhibitory activity of the recombinant chimeric plasminogen activator to thrombin in vitro .