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Objective To construct the eukaryotic expression vectors of interleukin-12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF), PBI-CMV3-IL-12 and PBI-CMV3-GM-CSF, and the expression of IL-12 and GM-CSF in hepatoma cells after H22 hepatoma cells are transfected with such vectors. Methods The Trizol method was used to extract total liver RNA from mice, which was then reversely transcribed into cDNA. The coding sequences of IL-12 and GM-CSF were obtained by amplification using specific primers containing BamHI and HindIII restriction sites. Double enzyme digestion was performed for the products and PBI-CMV3 empty vectors, and then the digested products were transformed into DH5α after being recycled by gel extraction kit and connected by T4 DNA ligase. Monoclonal bacterial colonies were selected and plasmid extraction, enzyme digestion, and DNA sequencing were performed for the identification of the expression vector constructed. The constructed plasmids were divided into empty vector group, PBI-CMV3-IL-12 group, PBI-CMV3-GM-CSF group, and co-transfection group. These plasmids were transfected into H22 hepatoma cells, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of IL-12 and GM-CSF in cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett′s T3 method was used for further comparison between two groups. Results The eukaryotic expression vectors, PBI-CMV3-IL-12 and PBI-CMV3-GM-CSF, were successfully constructed. Quantitative real-time PCR showed that there were significant differences in the mRNA expression of IL-12 and GM-CSF between the four groups (F=522 and 163, both P＜0.001). Compared with the empty vector group, the PBI-CMV3-IL-12 group and the co-transfection group had a significant increase in the mRNA expression of IL-12 (both P＜0.05). Compared with the empty vector group, the PBI-CMV3-GM-CSF group and the co-transfection group had a significant increase in the mRNA expression of GM-CSF (both P＜0.05). Western blot showed that the interest protein could bind to antibody and produce a specific band in the membrane, and an analysis of band size and gray value showed that the protein expression of IL-12 and GM-CSF had a similar trend as the mRNA expression of IL-12 and GM-CSF. Conclusion The eukaryotic expression vectors of IL-12 and GM-CSF are successfully constructed, and they are highly expressed in H22 hepatoma cells. [ABSTRACT FROM AUTHOR]