Construction and identification of the recombinant lentiviral vector expressing human bFGF gene

Objective To construct a recombinant lentiviral vector expressing human basic fibroblast growth factor (bFGF) gene. Methods Huamn bFGF and FGF4 genes were amplified by polymerase chain reaction (PCR) technique and then cloned into the pGC-FU vector which contained green fluorescent protein (GFP). The resulting lentiviral vector containing FGF4 and bFGF was named the plasmid of pGC FU-FGF4-bFGF vector. The correct bFGF gene was confirmed by PCR, endoenzyme digestion, sequencing analysis and contrast. The plasmid of pGC FU-FGF4-bFGF vector was co-transfected together with lentivirus-packaging plasmid pHelper 1.0 and pHelper 2. 0 into 293T packaging cells by lipofectamine 2000 mediation. The newly constructed recombinant lentivirus and the titer of virus were confirmed by real-time quantitative PCR. Results The 500 bp DNA sequence showed that the cloned bFGF gene sequence was the same as that of the published sequence. The evidence of endonuclease digestion, DNA sequencing and PCR analysis confirmed that bFGF gene was correctly inserted into the lentiviral vector, and the titer of rirus was 2.0 × 109 TU/ml. Conclusion The recombinant lentivirus expressing human FGF4 and bFGF were successfully constructed and effectively expressed in 293T cells, which probably can provide a reliable tool for genetic transfection in further gene therapy researches. Key words: bFGF;  Lentiviral vector;  Gene transfection