Construction Of Recombined Human EIF4E-shRNA Lentivirus And The Preliminary Research Of Its Infection To Hela Cell Line

Objective To study the silencing effects of eIF4E on the gene therapy of cervical cancer, in this study, we designed the small interference RNA (siRNA) specific to human eIF4E gene by RNA interfering technique, constructed its recombinant lentiviral expression vectors, and identified these vectors by PCR and sequencing. The packaging cells 293T were transduced by recombinant lentiviral vectors and the relevant supernatant was collected and concentrated and titrated. Hela cells were then infected by lentivirus and observed preliminarily.Methods Short hairpin RNA targeting to eIF4E was desigened and cloned into lentivirus work vector pLVTHM which was linearized by restriction endonucleases. The recombined vectors were transformed into competent E.coli.TOP10 cells. The positive recombinant colony was selected by ampicillin medium agar and identified by PCR and sequencing. The recombinant vector and three package plasmids were cotransfected to 293T cells with LipoD293 DNA In Vitro. Lentivirus in the 293T media supernatant were collected and concentrated by centrifuging. Then Hela cells were infected by concentrated lentivirus and observed preliminarily.Results Four recombinant eIF4E shRNA lentivirus vectors were constructed successfully and identified by PCR and sequencing. The recombinant lentivirus was harvested and concentrated after 293T cells were cotransfected. The titer was 4×108 TU/ml. The recombinant lentivirus could efficiently infect Hela cells and the green fluorescent was detected.Conclusion Four recombinant eIF4E shRNA lentiviral vectors were successfully constructed and Hela cells were infected successfully by the relevant lentivirus. These results laid the foundations for the further study on the gene therapy of cervical cancer.