Construction of human Annexin A1 cDNA in a eukaryotic expression vector

Annexin A1 (ANX I) is an inducible protein and acts through inhibiting cPLA2a to block the release of arachidonic acid and its subsequent conversion to eicosanoids. It reduces the activity of iNOS and COX-2 enzymes. Therefore, this protein may be effective in prevention of cell inflammatory conditions. Thus to elucidate the protective effects of ANX І in neuroinflammtory disorders, we decided to construct its coding sequence (CDS) in an appropriate vector to examine its overexpression effects in neuroinflammatory conditions in a neural progenitor cell. Hence, at first step, RNA was extracted form fresh blood sample taken from a healthy individual donor. cDNA was synthesized and RT-PCR was performed with suitable primers for human ANX I. In the second step, The amplified fragment was subsequently used again for PCR, to introduce AgeI Cleavage site and FLAG upstream and downstream of ANX I CDS, respectively. CDNA fragment encoding ANX I-Flag was inserted into the pTZ (T)-Vector. The recombinant plasmid was sequenced to ensure proper cloning of ANX I CDS. Finally, DNA fragment encoding ANX I-Flag was inserted into the PGL268, an appropriate eukaryotic expression vector, which is an episomal vector containing S/MAR sequences to retain this vector at episomal state. Chimeric cDNA encoding ANX I-Flag was appropriately inserted in an expression vector to examine its further applications in neural precursor cells. Due to the presence of S/MAR sequences in the plasmid, the overexpression of ANX I would be in episomal state.