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.


CONCLUSIONS
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.
Lu
Yang
Y
International Joint Cancer Institute & Eastern Hospital of Hepatobiliary Surgery, Second Military Medical University, Shanghai 200433, China.
Xue
Qi
Q
Qian
Wei-Zhu
WZ
Wang
Hao
H
Wu
Meng-Chao
MC
Guo
Ya-Jun
YJ
D000818:Animals; D019496:Cancer Vaccines / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D007158:Immunologic Techniques; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000235:genetics; D019076:Transgenes; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000819:agonists* / Q000235:genetics
D019496:Cancer Vaccines; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D040281:Vascular Endothelial Growth Factor Receptor-1