Establishment of vascular endothelial cell line stably expressing KSHV vIL-6 gene transduced by lentivirus

Objective To establish a vascular endothelial cell line stably expressing virus IL-6(vIL-6) gene transduced by lentivirus.Methods The vIL-6 gene was amplified from plasmid pvIL-6-Flag by PCR.The purified vIL-6 gene fragment was inserted into lentivirus vector(p3DLV) and the recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing.293T cells were cotransfected with the recombinant plasmid and the helper plasmids,thus the recombinant lentivirus carrying vIL-6 was successfully packaged.The recombinant lentivirus was transfected the vascular endothelial cells EA.hy926,and the resistant cell clones were selected with hygromycin.The expression of vIL-6 was examined using western blot.Results Enzyme digestion and DNA sequencing showed that the full-length vIL-6(615 bp) gene was successfully subcloned into the lentiviral vector.After transfection,the recombinant lentivirus was packaged into 293T cells.The titer of recombinant lentivirus was 1.0×107 TU/mL.The monoclonal cell line EA.hy926-vIL-6 was produced after transfection with the recombinant lentivirus and selected with hygtomycin.The stable expression of vIL-6 protein was verified by western blot.Conclusion An EA.hy926 cell line stably transduced with lentivirus carrying vIL-6 gene was successfully generated.It can be used as a cell model to study the biological function and pathogenesis of vIL-6.