Objective: To construct a eukaryotic co-expression plasmid containing FUS1 and IL-12, and to investigate the influence of the recombinant plasmid on the cultured human lung cancer cell line A549.
Methods: RT-PCR was applied to amplify FUS1 from MRC-5 cell, the cDNA fragment of IL-12 was derived from pORF-hIL-12 by PCR, FUS1 cDNA fragment and IL-12 cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-FUS1-IL-12. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenceing and transfected into A549 cells. The expression of genes in pVITRO2-FUS1-IL-12 were identified by RT-PCR, ELISA and Western blot. The function of pVITRO2-FUS1-IL-12 inducing the apoptosis of lung cancer was identified by Hoechst33258 and flow cytometry.
Results: The restriction endonuclease digestion and sequencing suggested that co-expression vector pVITRO2-FUS1-IL-12 was constructed successfully, the expression of FUS1 and IL-12 could be detected in A549 cells. The expressions of the two genes in pVITRO2 were not affected. The A549 cells transfected with pVITRO2-FUS1-IL-12 exhibited significant cell apoptosis.
Conclusion: The recombinant eukaryotic expression vector pVITRO2-FUS1-IL-12 was constructed and expressed in eukaryotic cell successfully, which would contribute to a novel and effective treatment strategies for combined gene therapy for lung cancer.