Construction of murine interleukin-12 eukaryotic expression plasmid and its expression

OBJECTIVE: To construct the plasmid that can express murine IL-12 gene stablely in eukaryotic cell in order to provide basis for further study of its immunoregulatory mechanism and antitumor immune response with mIL-12. METHODS:Plasmid pORF-mIL-12(Elasti) was amplified through PCR,objective segment (mIL-12) was inserted into eukaryotic expression vector pcDNA3.1(+) after enzyme digestion with restriction enzyme HindⅢ and EcoRⅠ, and mIL-12 was drived by mCMV promoter of pcDNA3.1(+) and transcribed to the same mRNA. The recombinant plasmid was transfected into LLC cell line after identifying it and measure its expression. RESULTS:mIL-12 has been amplified and identifyed it through PCR, enzyme digestion and sequence analysing, and pcDNA3.1(+)-mIL-12 plasmid has been constructed successfully. RT-PCR and ELISA results confirmed that mIL-12 had high expression at mRNA and protein level. CONCLUSION:pcDNA3.1(+)-mIL-12 eukaryotic expression plasmid is constructed successfully and it can express murine IL-12 gene stablely in LLC cell line.