Objective: To clone the p40 and p35 subunit cDNA of porcine IL-12(pIL-12) and construct the fusion gene of recombinant porcine single-chain interleukin-12 (pscIL-12).
Methods: The total RNAs were extracted from porcine peripheral blood mononuclear cells (PBMCs) and porcine splenic lymphocytes for cloning pIL-12 p35 and p40 cDNA by RT-PCR. A hydrophobic polypeptide linker (Gly4Ser)3 was used for splicing two different gene fragments (pIL-12) p40+linker+p35 (pscIL-12) by recombinant PCR to construct pscIL-12 fusion gene. The pscIL-12 fusion gene was then inserted into pcDNA3.1(+) eukaryotic expression plasmid, and the resulted pcDNA3.1(+)-pscIL-12 was transfected into CHO-K1 cells via lipofectin. The expression of pscIL-12 mRNA in the transfected cells was identified by RT-PCR.
Results: The sequence of the cloned porcine IL-12 p40 and p35 cDNA and the constructed pscIL-12 fusion gene were verified by PCR, restriction enzyme digestion and sequencing. The mRNA of pscIL-12 fusion gene was detected in the transfected CHO-K1 cells by RT-PCR.
Conclusion: The constructed pcDNA3.1(+)-pscIL-12 eukaryotic expression plasmid allows expression of pscIL-12 in CHO-K1 cells, thus facilitating further study of the biological activity and adjuvant effect of pscIL-12 fusion protein.