Construction of the eukaryotic expression plasmid contaning the gag gene of HIV-I subtype B and its expression in HepG2 cells

To construct a eukaryotic expression plasmid containing the gag gene of HIV-1 subtype B and to detect its expression in HepG2 cells in order to lay the foundation for further development of DNA vaccine against HIV infection, a pair of primers were designed and synthesized according to the published sequence of gag gene in GenBank. After amplification with PCR, the amplified product was cloned into plasmid pIMD18-T using TA cloning followed by Bam HI and Xho I digestion and sequencing. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1(+), and the recombinant plasmid was sequenced and identified by restrictive endonuclease digestion later on, the constructed recombinant plasmid was finally transfected into HepG2 cell line by using the lipid transfection reagent ,and the expression of the gag gene was analyzed by RT-PCR and Western blotting. It had been demonstrated that the successful construction of the recombinant vector pcDNA3.1(+)/gag was verified by restrictive endonuclease assay and sequence analysis with the same target fragment gag as U26942 in GenBank, and the expression of the gag gene was detectable in the lysate of transfected HepG2 cells as demonstrated by RT-PCR and Western blotting. Thus it is concluded the eukaryotic expression/plasmid for gag gene is successfully constructed and its expression in HepG2 cell line was also confirmed in the present study.