Expression Of Fibrin (Ogen) Olytic Enzyme Gene From Gloydius Intermedius Venom

Nowadays thrombotic diseases are one of the most serious diseases that greatly affected people's life. Among all of the therapies, thrombolytic therapy is regarded as the most effective method to cure the thrombotic diseases. The fibrin(geno)olytic enzymes (FLE) which wildly exist in many kinds of snake venoms as a kind of novel powerful thrombolytic agent can directly lysis fibrin(ogen). FLE purified from natural snake venoms through the technology of protein separation display the potential value in the application of cardio-cerebrovascular disease. However, because of the reasons of natural snake venoms'limited resources and theirs difficult separation, storage and transportation, how to research and develop a native and effective thrombolytic with molecular biological methods and genetic engineering technology will play a significant and profound role in the antithrombotic processes.The FLE sequence, cloned from Gloydius intermedius venom gland, which was cutted by double digestion from pMD18T-FLE I plasmid was subcloned to pFASTBACHTa expression vector. Recombinant eukaryotic expression plasmid pFASTBACHTa-FLE was constructed after transformation, selection and identification. The recombinant plasmid was injected into mice in large quantity and large volume (20μg) in a short time (5s) through tail vein. These mice were sacrificed 8h after the injection. Their hepatic tissues were used to prepare the experiment samples.SDS-PAGE analysis showed that the recombinant protein in liver tissue has a molecular weight approximately 30kDa. Recombinant FLE were examined in liver tissue with Western blot which showed that there was a specific hybrid band. Immunohisto-chemistry showed that there were a great of recombinant FLE could be found in liver tissue. The fibrinolytic activity of recombinant protein revealed by the modified fibrin plate method demonstrated that the recombinant FLE in liver tissue was efficiently expressed and its thrombolysis was demonstrated to be dose-dependent and time-relative.As a result, FLE, recombinant protein, was obtained successfully from mice's hepatic tissue. It laid a solid foundation and prepared experimental material for the future studies on the bioactivity of snake venom FLE.