[Screening of the clones efficiently expressing rhIL-12 in CHO cells and functional analysis of the expressed rhIL-12].

AIM

To transfect the constructed recombinant human IL-12 (rhIL-12) eukaryotic expression plasmid (pcDNA6/v5-his-p70) into CHO cells and screen the efficiently and stably expressed clones; and to identify the biological activities of rhIL-12.

METHODS

pcDNA6/v5-his-p70 was transfected into CHO cells using polyethyleneimine (PEI) and positive clones were screened with Blasticidin as well as single clone amplification. Then the expression clone was identified by RT-PCR and the rhIL-12 expression of each clone was measured using ELISA. Finally, biological activities of the expressed rhIL-12 were analyzed with proliferation of lymphocytes in vitro and intracellular cytokine staining(ICS) . The stability of the selected clones was observed as well.

RESULTS

The RT-PCR results showed that a specific fragment of 1 800 bp could be amplified in all positive clones, among which six clones had rather high expression of 312.69 ng/L-719.10 ng/L with the highest expression of 719 ng/L (5x10(4) per cells, 48 h) by ELISA test. Biological activities analysis indicated that the expressed rhIL-12 could significantly increase the percentage of CD4(+)IFN-gamma(+) and CD8(+) IFN-gamma(+) cells in normal PBMC as well as the proliferation of PBMC to PHA/HCMV stimulation. The efficient expression of positive clones still remained stable after 6 months under maintenance of Blasticidin (2 ng/L).

CONCLUSION

The constructed human recombine hIL-12 eukaryotic expression vector can express stably with high efficiency in CHO cells and the expressed products show expected biological functions.