كلونينگ، بيان وتخليص فاكتور VII نوتركيب انساني به صورت فيوژن با برچسب هيستيديني ‏با استفاده ازفناوريGateway

Title

Expression and purification of recombinant human coagulation Factor VII fused to His-Tag through Gateway technology

English Abstract

Background. Factor VII is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. Construction, expression and purification of recombinant FVII fused to poly histidin tag through gateway technology were aimed in this study. Methods. To construct entry clone, blunt-end FVII cDNA and subsequent PCR product isolated from HepG2 cell line was TOPO cloned into pENTR TOPO vector. To construct expression clone, LR recombination reaction was carried out between entry clone and destination vector, pDEST26. CHO c ells were transfected with 1 g of DNA of PDEST26 FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant factor VII were established. The expression of recombinant FVII was confirmed by RT-PCR and ELISA. Culture medium containing his-FVII was added to the nickel-nitrilotriacetic acid resin c olumn and bound protein was eluted. The purified protein was detected by SDS-PAGE and western blot analysis. Biological activity of the recombinant factor VII was determined by prothrombin time assay using factor FVII-depleted plasma. Results. The results showed that human recombinant FVII successfully was cloned and accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but any expression was not detected in the CHO cells containing empty vector. A protein of about 52KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed by using this rFVII. Conclusion. As we are a ware, this is the first report of expression of recombinant FVII fused with his-tag through gateway technology. The next steps including large scale expression, purification, activation and stabilization are underwa y.

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