Construction of pGFP-FL plasmid and its application in preparing FL-secreting tumor vaccine.

OBJECTIVE: To construct a plasmid containing a green fluorescent protein (GFP) reporter gene as the effective vector for preparing fms-like tyrosine kinase receptor-3 ligand (FL)-secreting tumor vaccines. METHODS: A pGFP-FL plasmid, harboring a FL gene and a GFP gene, was designed and constructed by routine molecular cloning techniques. In this plasmid, FL gene was under the control of cytomegalovirus promoter, while EF-1a promoter acted to drive GFP gene. A prokaryotic/eukaryotic selective gene Kan(R)/neo was also introduced into the plasmid. After structure identification by restriction analysis, pGFP-FL plasmid was further transferred into Hepa1-6 cells, and the expression of GFP and FL genes was examined by way of fluorescent microscopy and reverse transcriptase-PCR respectively. RESULTS: Restriction analysis showed that the structure of pGFP-FL plasmid was exactly the same as anticipated. Further results indicated that both GFP and FL genes were simultaneously expressed in Hepa1-6 cells. CONCLUSION: A new plasmid has been established as the vector for studying the FL-secreting tumor vaccines, in which GFP gene can serve as a reporter gene reflecting the expression of FL gene.