[Construction and expression of the eukaryotic plasmids containing different subunits of human IL-12 gene].

AIM

To clone and construct the eukaryotic expression plasmids containing different subunits of human IL-12 and to investigate its expression following transient transfection into COS-7 cells.

METHODS

Human leukaemia cells (HL-60 and THP-1) were stimulated by DMSO, IFN-gamma and LPS, and the target genes including IL-12 p40 and p35 subunits were amplified by RT-PCR. P40 and p35 fragments were then connected and the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 were constructed by double restriction enzymes digestion and connection. Positive plasmids were transiently transfected into COS-7 cells with siPORT XP-1 reagent. The expression of target gene was detected by RT-PCR and ELISA.

RESULTS

The recombinant plasmids were confirmed by PCR amplification, double restriction enzymes digestion and DNA sequencing. Target gene expression was detected by RT-PCR and ELISA in the transfected COS-7 cells.

CONCLUSION

The successful construction of the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 provides a useful tool for further research on the immune regulatory role of IL-12.