Study on Construction of the homologous recombinant adenovirus tk gene

Objective To explore the possibility of a high titer of the Recombinant Adenovirus Adeasy System induced Hsv-tk gene. Methods The KDR-tk fragment were taken from pBluescript II KDR-tk plasmid, then the fragment of shuttle vector ptrack and ptrack CMV in Adeasy system and tk fragment were ligated according to the instruction of Takara DNA Ligation Kit. The green fluorescence protein (GFP) positive stained cells was counted under fluorescent microscopy. Results The PCR and the RT-PCR results both indicated that the virus induced tk gene was combined in the gene of recombined adenovirus gene. It suggested that tk gene could be expressed effectively in DNA and mRNA. The GFP positive stained cells were counted,and the titer of the virus came to be 1.1 × 1011 pfu/L. Conclusion The pAdeasy System may be used to produce a high titer of the recombinant adenovirus induced tk gene. The GFP expressing kit may be used directly to observe the transfection efficacy under fluorescent microscopy.