Construction of human single chain interleukin-12 fusion gene by recombinant PCR

A hydrophobic polypeptide linker (Gly_(4)Ser)_(3) was used to splice two different gene fragments of p40 and p35 subunits of hIL-12 (hIL-12 p40-linker-p35) by recombinant PCR for constructing rhscIL-12 fusion gene in vitro. After sequencing, rhscIL-12 fusion gene was inserted into pcDNA3.1(+) eukaryotic expressing plasmid and then transfected into COS-7 cell by lipofection. The fusion protein expressed was analysed with Western blot. Sequence analysis showed that the splicing order, the direction and the sequence in hscIL-12 fusion gene were completely correct. Expressed hscIL-12 fusion protein in COS-7 cells combined with mouse-anti-human IL-12 monoclonal antibody specifically. Western blot showed the apearance of 70 000u band of fusion protein. The result indicates that recombinant PCR is a very effective and reliable method for gene recombination in vitro.