Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells
Abstract: Full-length NS5A gene of Hepatitis C virus（HCV） was amplified by PCR, using the plasmid pBRTM/HCV 1-3011 containing HCV full-length open reading frame（ORF）as template, and cloned into the eukaryotic expressing plasmid pcDNA3.1（-）by DNA recombination technique. The recombin- ant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.1（-）-NS5A and the control vector pcDNA3.1（-）were transfected Hela cells using LipoVecTM. The cells expressing NS5A stablely were selected by G-418 and further proved by RT-PCR and Western blot analysis. We found the growth of Hela cells expressing NS5A was slower than the cells transfected by pcDNA3.1（-）in the same culture condition, and the population doubling time of Hela cells expressing NS5A gene is increased about 50%(about 35-35 hours). There was no significant difference between the control cells and the cells transfected with pcDNA3.1(-) (about 23-24 hours). The results indicate that NS5A can inhibit the proliferation of Hela cells.