Construction of HeLa Cell Lines Stably Expressing HCV 1a NS5A and NS5A-ΔISDR

The gene fragments of ns5a, LISDR and RISDR were amplified from pBRTM/HCV-3011. The ns5a was cloned into XhoI and EcoRI sites of pEGFP-N3 to construct pEGFP-N3-ns5a expressing vector, and LISDR and RISDR were sequentially cloned into XhoI/HindIII and HindIII/EcoRI sites of pEGFP-N3 to construct pEGFP-N3-ΔISDR(ISDR deletion). Then the recombinant eukaryotic expression vectors were identified by enzyme digestion analysis and sequencing. HeLa cells were transfected by electroporation with the two recombinant vectors. Cells were grown for 36 h in the standard media used to propagated each cell lines, after which G418 was added to 400 μg/mL to select for plasmid-bearing cells. HeLa clones expressing target genes were derived by limiting dilution at a concentration of 0.3 cells per well. The expression of NS5A and NS5A-ΔISDR in transfected HeLa cells were verified by RT-PCR and fluorescent microscope. Enzyme digestion analysis and DNA sequencing results showed that target genes have been cloned into the eukaryotic expression vector pEGFP-N3. The expression of NS5A and NS5A-ΔISDR have been demonstrated by RT-PCR and fluorescent microscope. The recombinant eukaryotic expression vectors have been constructed and expressed successfully in the transfected HeLa cells. It is possible to use HeLa cell lines stably expressing HCV NS5A and NS5A-ΔISDR to detect whether the resistance to interferon therapy of some HCV genotypes is related to ns5a.