Construction of a pFast Plasmid-based Expression Vector for Expression and Purification of Human Blood Coagulation Factor VIII, the Causative Agent of Hemophilia A
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Hemophilia A is an X-linked recessive genetic disorder caused by mutations in the gene coding for the Factor VIII (F8). Since the F8 protein is a vital component of the blood coagulation cascade, mutations in the F8 gene lead to a functionally deficient F8 protein that inhibits a patient?s ability to produce adequate blood clots. This deficiency in F8 activity induces life-threatening and uncontrollable external and internal bleeding. The current treatment for hemophilia consists of injections of blood serum from healthy humans, which becomes increasingly ineffective over time and carries with it the risk of infection by HIV and other autoimmune diseases. The main goal of this summer?s research was to construct a plasmid vector housing functional F8-coding gene that could eventually be used to produce a functional protein in insect cells. To do this, we conducted a series of restriction digest and ligation reactions as well as sequencing of the final DNA construct. Future work on this project will focus on production and purification of recombinant F8 protein from insect cells. This purified protein can then be used to develop a treatment procedure for hemophiliacs in which the F8 is injected intravenously, which would eliminate the risk of a built-up immunity or inadvertent infection.