Expression And Identification Of Recombinant Human Coagulation Factor Ⅶa In Eukaryotic Cells

Human blood coagulation factor â…¦(hFâ…¦) is a vitamin K-dependent, single-chain glycoprotein that participates in the extrinsic phase of blood coagulation. Recombinant activated factor â…¦ (rFâ…¦a) initiates the coagulation cascade by binding to tissue factor at the site of injury and causes the formation of sufficient amounts of thrombin to trigger coagulation. rFâ…¦a was sanctioned into market in 1999 by FDA (NovoSeven. rFâ…¦a and pdFâ…¦a (derived from human plasma) share the identical function though several differences exist in the structure between them. Patients with a variety of other coagulation deficiencies than hemophilia characterized by an impaired thrombin generation and life-threatening bleeding have been reported as successfully treated with rFâ…¦a. Due to the safety and effectivity, rFâ…¦a is expected to be helpful to save lives in the war.To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding human factor â…¦ was isolated using polymerase chain reaction-mediated DNA amplification from a plasmid pUC19 in which F7 was inserted. Human factor â…¦ cDNA was transfected into two separate cell lines (BHK and CHO) and cell lines that permanently expressed human recombinant factor â…¦ was established. The expression product was concentrated from tissue culture media using a millipore filtration tube. The purity and authenticity of human recombinant factor â…¦ was confirmed by polyacrylamide gel electrophoresis and Western blot analysis. Homogeneous human recombinant factor â…¦ was fully active biologically as determined by prothrombin time assay in factor Fâ…¦ -depleted plasmas, human thromboplastin was responsible for the activation process, in which Fâ…¦ broke into two fragments, as showed in SDS-PAGE. Human recombinantfactor â…¦ should prove useful for future in vivo investigations of experimental coagulopathies.In another experiment, a new expression vector was constructed in order to transfect Pichia pastoris. We detected the existence of Fâ…¦ in the culture media as determined by SDS-PAGE and western blot, yet we did not find any biology activity, which indicated that Pichia pastoris was not suitable for Fâ…¦ expression.