In vitro transcription of eIF4E in laryngeal carcinoma
Wen Lian-ji · Chinese Journal of Otorhinolaryngology-skull Base Surgery · 2008
Objective To clone the cDNA sequence of eIF4E coding area in Hep-2 cell,to construct its transcriptive vector,and transcript the eIF4E mRNA in vitro.Methods Using Hep-2 cell cDNA as template,full-length eIF4E was obtained with the technique of RT-PCR followed by automatic sequencing of pMD18T-eIF4E,and its in vitro transcription plasmid pGEM-7zf+eIF4E was constructed with recombinant DNA technique.Then pGEM-7zf+eIF4E plasimid was transcripted in vitro with T7 trascription kit,and eIF4E mRNA was labelled with [a-32P]UTP.Results Sequencing proved that the cloned eIF4E cDNA was completely identical with that of Gene Bank,and the recombinant in vitro transcriptive plasmid pGEM-7zf+eIF4E was constructed successfully.Conclusion eIF4E cDNA is successfully cloned and an in vitro transcriptive plasmid pGEM-7zf+eIF4E is constructed.