Chicken Oviduct-specific Expressing Of Recombinant Human Coagulation Factor Ⅸ

hFIX (human coagulation factor IX) is a vitamin K-dependent coagulation factor and the precursor of a serine protease required for blood clotting triggered by the intrinsic clotting pathway, and thus plays an important role in the intrinsic clotting pathway. Hemophilia B is a sex-linked clotting disorder due to deficiency of hFIX activity that affects1of30000males. The major clinical manifestations of these patients were blood clotting disorder after slight bleeding or surgical bleeding, even spontaneous bleeding. Human PCC (prothrombin complex concentrates) is the most common blood product for clinical treatment of hemophilia B. The supply of PCC always falls short of demand because of the shortage of fresh plasma. Therefore, it is very necessary to develop a bioreactor to produce high quality of hFIX for clinical use at low cost using molecular biology technique and biological engineering tools. It will not only be a gospel to all the patients with hemophilia B, but also have a great potential in medical market.The purpose of this study was to build a recombinant plasmid vector that could specifically express hFIX protein in the oviduct of laying hens as an ideal bioreactor, so that hFIX protein could be secreted into eggs and extracted from the eggs for clinical use.The experimental procedures used in this study were described as follows:to get the recombinant hFIX gene sequence that could be expressed in laying hens, we optimized the codon of hFIX gene using the published hFIX gene sequence on NCBI (Genebank accession number: M11309.1), the preferential codon usage of chicken, and bioinformatics software. The cDNA of the recombinant hFIX gene was then synthesized by a biotechnology company. The cDNA was subsequently cloned into a plasmid vector (pL-OM) that contained the promoter of ovalbumin gene to build a construct called pL-hFⅨ, which could drive the expression of the recombinant hFⅨ specifically in chicken oviduct. The pL-hFⅨ vector was transferred into DH5a competent cells, followed by cell culture and plasmid DNA extraction. The purified DNA was then mixed with gene transfection reagents-PEI and injected into wing vein of laying hens. During the experiment, the eggs were collected and egg whites were isolated from the eggs and examined for hFⅨ expression by ELISA analysis.The results of this study indicated that the hFⅨ protein could be detected in3of the10tested laying hens.The concentration of hFⅨ protein in egg whites was in a trend of increase ranging from108to144U/L. The results suggest that the expression vector pL-hFⅨ is correctly constructed allowing hFⅨ protein successfully and specifically expressed in the oviduct off laying hens.The significance of this study:we built an oviduct specific expression system for hFⅨ protein that allows for a high level of expression in egg whites of laying hens. It may provide us a foundation for the production of high quality of hFⅨ protein at low cost using chicken oviduct as a bioreactor. This study may set up a guideline for similar research work..