OBJECTIVETo increase prokaryotic expression level of IFN-alpha 1C gene through the quantitative theory of translational control and the translational enhancer sequence.

METHODSStepwise polymerase chain reaction (PCR) was used to alter the 5 terminal cDNA sequence of IFN-alpha 1C in three different grades of base mutation. In this way, the free energy (Delta G) of the secondary structure in translational initiation region (TIR) was decreased gradually. An expression plasmid (pBVE) was constructed to contain the translational enhancer cDNA sequence by modifying pBV220 upstream of the SD region.

RESULTSThe expression levels of three kinds of IFN-alpha 1C modified gene were all increased. Furthermore, it presented an increasing trend with decreasing in delta G varying from -50,241.6 to -22,190.0 J/mol. The highest expression was 2.43 x 10(8) U/L, covering twelve times more than its original cDNA. IFN-alpha 1C gene and its modified cDNA was inserted into pBVE as reporting genes. E.Coli cells harbouring pBVE/IFN-alpha 1Cs cDNA produced two to five times more IFN than cells harbouring pBV220/IFN-alpha 1Cs.

CONCLUSIONSpBVE containing translational enhancer is a high level prokaryotic expression vector. The theory of quantitative translational control can effectively be used to enhance the IFN-alpha 1C gene expression level in E.coli.

DNA, Circular ; genetics ; Enhancer Elements, Genetic ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation ; Genetic Vectors ; Interferon Type I ; genetics ; Peptide Chain Initiation, Translational ; genetics ; Polymerase Chain Reaction ; methods