In silico investigation and synchronous application of introns 1 and 2 of human beta-globin to increase the expression of human coagulation Factor IX

Manipulation of regulatory elements is usually considered as a major act for the improvement of production of recombinant proteins. In this regard introns with the potential of regulatory function have provided useful means. Human beta globin gene has two introns with 130 and 850 bp lengths, in silco analysis of these two introns show that they carry a number of potential regulatory elements which make them useful to be applied for the expression of recombinant proteins in mammalian host cells. With the aim of the production of recombinant human factor IX (hFIX), a hFIX minigene carrying the human beta globin introns (hBGIs) was inserted in a CMV regulated vector. In the constructed vector the hBGIs was inserted in the corresponding position in hFIX-cDNA. In a separate line an intron-less hFIX-cDNA was inserted in a similar vector to be used as negative control. Chinese Hamster Ovary (CHO) cells were transfected with the constructed plasmids, based on lipofectoin method. The culture media taken from the transfected cells were examined for coagulation activity with a single stage clotting test performed on FIX-deficient plasma. The data obtained from the expression analysis of the two groups of transfected cells in comparison with the cells with the intron-less cDNA indicates in the expression of hFIX by both groups of cells. The same data shows a relatively higher coagulation activity in the culture media taken from the cells with intron-containing hFIX-cDNA.