Prokaryotic Expression Of SA-hirudin-RGD Fusion Proteinand Identification Of Its Anticoagulant And Anti-platelet Aggregation Function

Objective: To construct a recombinant vector SA-hirudin-RGD expressing purified fusion protein, and to preliminary validate its anticoagulant and anti-platelet aggregation function.Methods: The natu streptavidin(SA) ral core sequence and hirudin-RGD gene were cloned into pET-44 b to form the recombinant plasmid pET44b-SA-hirudin-RGD. The fusion protein was induced by IPTG, purified by Ni-NTA agarose and detected by Western-blot. The anticoagulant and anti-platelet aggregation assay to prove the activity of SA-hirudin-RGD.Results: Restriction enzyme digestion and gene sequencing showed that the recombinant plasmid pET44b-SA-Hirudin-RGD was successfully constructed. The fusion protein induced by IPTG was efficiently expressed in E.coli. Fusion protein was purified by Ni-NTA agarose with a molecular weight of 70000 Da identified by Western blot. The assays proved that SA-hirudin-RGD has anticoagulant and anti-platelet aggregation activity.Conclusion :SA-hirudin-RGD fusion protein with anticoagulant and anti-platelet aggregation function is successfully expressed and purified, which lying a good basis for further functional and clinical application of SA-hirudin-RGD.