Cloning and expression of recombinant tissuefactor in CHO cells

Background and ObjectivesTissue factor (TF), a 45-kDa transmembrane glycoprotein, is the major cellular initiator of the coagulation cascade. Prothrombin Time (PT) is the test that evaluates extrinsic pathway of coagulation. Thromboplastin used in this test is mostly prepared of rabbit brain. Thus it causes variation in the PT results. There is an important advantage of using recombinant TF in PT test to get more reproducible results. Materials and MethodsTF mRNA was isolated from human lung fibroblast cells. After preparation of cDNA it has cloned in pcDNA3 plasmid. CHO cells were transfected with recombinant plasmid. Transfected cells were grown in presence of Geneticin. Total proteins were extracted from CHO cells and separated with SDS/PAGE electrophoresis and analyzed by western blotting technique. CHO cells expressing TF were added to citrated plasma in presence of Cacl2 and clotting time was measured. In addition, factor VII activation by recombinant TF in the plasma was assessed by ELISA method. ResultsExtracted proteins from tarnsfected CHO cells separated on SDS/PAGE showed an approximately 40-kDa band that verified with a monoclonal antibody against TF on western blot analysis. Adding transfected cells (106cell/ml) to the citrated plasma in presence of Cacl2 could decrease clotting time from 3 minutes to 21 ± 3 seconds compared with untransfected CHO cells. The level of factor VIIa in citrated plasma was 810 ± 35 ng/ml in presence of transfected cells’ membrane.ConclusionsRecombinant TF expressed in CHO cells was able to clot citrated plasma and to activate factor VII.