Construction of cDNAs for A1-A2 and A3-C1-C2 Domains of Factor VIII (Δ B, 3 and 5 UTR) by RT-CPR

Background-Construction of complementary DNA (cDNA) is the first step to produce a recombinant protein and reverse transcription of mRNA is the best way to construct cDNA. Construction of cDNAs for biologically active domains of factor VIII has advantages of joining two domains to form a second generation of factor VIII and expression of domains separately for especial purposes.Method-Total RNA was purified from HepG2 and 293 cell lines, were quantitated and analyzed for presence of factor VIII mRNA by Dot RNA Blotting. One and two step reverse transcription polymerase chain reaction (RT-PCR) were applied. Primers (including nested primers) were designed for both sides of A1-A2 (cDNA1) and A3-C1-C2 (cDNA2) domains with respect to saving open reading frame (ORF) of cDNAs and deleting of B-domain, 3` and 5` untranslated region (UTR). Restriction mapping and sequencing were applied for analysis and confirmation. cDNAs were cloned into the vector and then into the ultracompetent cells by electroporation. Plasmids were purified, restriction analyzed and sequenced.Results-Five clones of cDNA1 and seven clones of cDNA2 were produced and checked for sequence similarity with factor VIII sequence from database. Some of them had point mutations and were suitable for the assessment of biological activity and yield of production.Conclusion-The construction of cDNA for A1-A2 and A3- C1-C2 domains and cloning of these two domains, which will be later linked with metal ions, is a way for production of second generation recombinant factor VIII. Another application for recombinant domains would be immune tolerance induction in hemophiliacs with inhibitors against these domains without need for administration of whole molecules.