[The expression of human IL-1β gene containing human erythropoietin signal peptide in HepG2 cells].

OBJECTIVE

To construct two lentiviruses secreting human IL-1β through either classical or nonclassical pathway and analyze their expressions in HepG2 cells after packaging lentiviruses and infecting hepatoma carcinoma HepG2 cells.

METHODS

Human full-length IL-1β gene and chimeric gene containing human erythropoietin(EPO) signal peptide and mature IL-1β protein coding sequence were respectively amplified from pIRES2-EGFP-proIL-1β and pIRES2-EGFP-epoIL-1β using PCR. The sequences were subsequently cloned into lentiviral expression vector pLenti6/V5 to construct pLenti6/V5-proIL-1β, which expressed IL-1β through nonclassical pathway, and pLenti6/V5-epoIL-1β, which expressed IL-1β through signal-peptide mediated classical pathway. Lentiviruses expressing human IL-1β through either classical or nonclassical pathway were packaged in HEK293T cells using a three-plasmid packaging system, and then these viruses were used to infect HepG2 cells. The level of IL-1β in both cytoplasm and culture supernatant were detected by sandwich ELISA and Western blotting.

RESULTS

pLenti6/V5-proIL-1β expressing human full-length IL-1β gene and pLenti6/V5-epoIL-1β expressing human EPO signal peptide and mature IL-1β gene were successfully constructed and confirmed through enzymatic assay and DNA sequencing. The lentiviruses expressing IL-1β through different pathways were prepared using a three-plasmid packaging system in HEK293T cells. Compared with the cells infected with control virus, levels of supernatant and cytoplasmic IL-1β in the cells infected with two lentiviruses expressing IL-1β through different pathways were markedly elevated (P<0.01). However, level of mature IL-1β in supernatant of HepG2/epoIL-1β cells was much higher than that of HepG2/proIL-1β cells, while total IL-1β level in cytoplasm of HepG2/proIL-1β cells was significantly higher than that in HepG2/epoIL-1β cells.

CONCLUSION

Both classical and nonclassical pathway secretion vectors could express human IL-1β in HepG2 cells, but EPO signal-peptide mediated classical pathway secreted much higher mature IL-1β than that of nonclassical pathway.