Construction And Overexpression Of Human INF-γ And CD In E.coli With Two-Cistron Expression System

The gene encoding human interferon-γ (IFN-γ) was cut with EcoR V Sal I and then subcloned into the same enzyme digested pGI vector for expression in Escherichia coli, which named pGIFN, and then was transformed into E.coli JF1125 .High expression efficiency of this system was confirmed by the SDS-PAGE optical density scanning result of which the rIFN-γ protein production could reach over 58% of the total bacterial protein following temperature induction. Another prokaryotic expression vector pGCD for cytosine deaminase(CD) was also constructed by inserting the PCR amplified product, E.coli HB101 CD gene, with Shine-Dalgarno(SD) sequence at its 5' end into pGI. The pGCD vector expressed recombinant CD protein at a high level over 47% of the total bacterial protein evaluated by SDS-PAGE. Based on the above results, the IFN-γ cDNA fragment was inserted into between EcoR I and Sma I restriction sites juxtaposing ahead the CD gene in pGCD plasmid to constructed a two-cistron expression vector pGIFN-CD. After transformed into E.coli JF1125 and induced by temperature, pGIFN-CD simultaneously expressed rIFN-γ and rCD protein with high efficiency that the IFN-γ and CD accounted for over 33% and 35% of the total bacterial protein, respectively. The research work laid the foundation for the combinational antitumor gene therapy using IFN-y and CD.