Expression And Activity Assays Of Prourokinase (144-411)-AnnexinV Chimera In Bombyx Mori Baculovirus Expression Vector System

Thrombolytic therapy has been a major advance in the treatment of myocardial infarction over the last decade. However, available thrombolytic agents have certain limitations. The thrombus may be resistant to lysis in some patients or may reform after initial lysis; and bleeding, particularly intracerebral, is a serious side effect. This has led to improve the potency and safety of thrombolytic agents. One approach has been to target the thrombolytic agent by preparing chimeric proteins in which a thrombus-specific antibody is attached to a plasminogen activator (usually a form of urokinase). Antibodies against fibrin and platelet-specific antigens such as glycoprotein Ilb/IIIa, and thrombospondin have been used.Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. A chimera was disigned that annexin V as a mean to target thrombolytic agents to platelet-containing thrombosis: prourokinase (144-411)-annexin V. To keep the accurate three-dimensional structures of the chimera, a 12-amino acid polypeptide of hirudin, a natural inhibitor of coagulation, was used as a linker between prourokinase (144-411) and annexin V.Prourokinase (144-411)-Annexin V Chimeras fuse gene was inserted into Bombyx mori baculovirus transfer vector pBacPAKS and co-transfected with lineared DNA of Bm-PAK6 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 and Western blotting analysis. The BmN culture cells and the fifth instars larvae were infected by recombinant baculovirus, BacPAK-UKAV, with 10pfu/cell. The biological activity of the protein product was detemined by APTT and fibrin-well assays. The yield of chimera from BmN cells was 100 U/106 infected cells. The results showed the inhibitory activity of the recombinant chimeric plasminogen activator to thrombin in vitro.The immunogenicity of insect cell-derived proteins, due to differeces in glycosylation patterns, and hence their usefulness as therapeutics remains to be determined.