Construction and Expression of a New Modified Coagulation FVIII cDNA In NIH3T3, CHO, and HepG2 Cell Lines

Introduction

Hemophilia A or factor VIII deficiency is a common X-linked genetic bleeding disorder in humans, occurring in about 10-20 males per million. For those afflicted, it is potentially life threatening and crippling.

Abstract

Hemophilia A or factor VIII deficiency is a common X-linked genetic bleeding disorder in humans. The FVIII contains a domain sequence organization designated A1-A2-B-A3-C1-C2, which the B-domain is not necessary in coagulation activity. We constructed a new B-domain deleted FVIII cDNA and cloned it into the N-terminal His tagged expression vector via the Gateway technology. This vector transfected into three cell lines: NIH3T3, CHO, and HepG2. The rFVIII extracted was purified and detected with SDS PAGE and western blot using anti-His tag and anti-FVIII antibodies and the rFVIII activity was measured using the ST4 kit. The results showed high expression and activity in NIH3T3 and CHO cell lines. The B-domain containing glycosylation sites was removed in this construct. As a result of this diminished glycosylation heterogeneity, B-domain truncated variants of FVIII may also be a more suitable precursor for making well-characterized, long-acting FVIII variants.

Keywords

blood coagulation factors, factor VIII, cloning, expression, gateway technology