Fig. 4

Establishment of HCV4a-HEK293T cell line for stable expression of HCV4a structural proteins. a Development of HCV4a-HEK293T stable cells. To develop a stable cell line for stable expression of HCV structural proteins, HEK293T cells were cultured and infected with the HCV4a-Lentiviruses. To isolate and enrich the HCV4a-HEK293T stable cells, the infected cells were maintained in medium containing puromycin (1 μg/ml) for 4–5 days to get rid of the uninfected cells before replacing the medium with fresh complete medium without the antibiotic for cells recovery. The expression of HCV core protein in the stable cells was investigated by IF using the HCV core antigen mouse monoclonal antibodies (C7-50) and the goat anti-mouse secondary antibodies conjugated with Alexa Fluor-555. Nuclei were counter-stained with the Hoechst 33342 blue stain. Un-infected cells were employed as a negative control for the core expression. b The integration of HCV genes into the genome of the established HCV4a-HEK293T stable cells. The total genomic DNA was isolated from the control and stable cells and subjected to the PCR analysis for detection of 1396 pb band of HCV4a Core-E2 genes using specific primers, HCV4a-RT-core-F and HCV4a-RT–E2–R. The β-actin gene was amplified using human β-Actin-F and Human β-Actin-R primers as a control. c Expression of HCV structural genes by stable cells. The mRNA expression of HCV integrated genes, Core, E1 and E2, in the HCV4a-HEK293T stable cells was examined by the isolation of the total RNA from normal and stable cells and used as templates to synthesize cDNA for both samples. The cDNA was subjected to the PCR analysis using specific primers for HCV4a Core, E1 and E2. The PCR for the β-actin gene from both cDNA samples was used as a control for the gel loading, while the PCR of β-actin gene using the RNA of both samples as a template was used as a control for detecting the DNA contamination in the RNA samples