Construction of the recombinant adenovirus ad-hil-6,and the express ion in euearyotic cell.

Objective To construct the adenoviral vector that expresses fusion gene encoding Hyper-IL-6 on the basis of successfully amplifying Hyper-IL-6 by PCR.Detect the expression of Hyper-IL-6 in the human hepatic cell lines LO2 treated with Ad-HIL-6.Methods The Hyper-IL-6 gene was coloned to the shuttle plasmid pAdTrack-CMV,The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells.Before transfection,the recombinant adenoviral plasmids were digested with PacI.Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000.Generation of recombinant adenovirus was monitored by GFP expression.Presence of recombinant adenoviruses was further confirmed by PCR.The expression of fusion gene encoding Hyper-IL-6 was detected in the Ad-HIL-6 treated LO2 cells by RT-PCR and western blot.Results The results show that we have successfully coloned the fusion gene encoding Hyper-IL-6 to the shuttle plasmid pAdTrack-CMV.The restriction analysis and the PCR conformed that correct recombinant adenoviral plasmid was constructed.Two days after transfection,the fluorescence was observed in 293 cells.the mRNA and protein of Hyper-IL-6 are successfully detected in the Ad-HIL-6 treated LO2 cells.Conclusion The success in construction of the adenoviral vector and expression of Hyper-IL-6 in LO2 cells make it possible to study further on Hyper-IL-6 mediated gene therapy.