Stable expression of T7 RNA polymerase mediated by lentivirus in MDBK cells

The low efficiency of transcripts in vitro mediated virus rescue seriously restricts the development and application of reverse genetics technology. t7 rna polymerase (rnap),a member of a family of single-subunit rnaps that includes the phage rnaps,transcribes the single or double-strand dna at the downstream of t7 promoter and subsequently synthesizes rna which complements with its corresponding dna template,and is extensively applied to viruses rescue in vivo. in order to establish a novel and efficient system for bovine viral diarrhea virus (bvdv) rescue,we successfully established a madin-darby bovine kidney (mdbk) -t7 cell line which stably expresses t7 rnap. in this study,we amplified the coding gene of t7 rnap from escherichia coli (e. coli) de3 (bl21) genome. after cloning into a prokaryotic expression vector pet-32a,the protein of t7 rnap was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). meanwhile,lentivirus was packaged with the recombinant lentiviral vector plex-t7 rnap and its corresponding helper plasmids. after lentivirus infection and puromycin selection,the drug-resistant cells were obtained. to identify the validity of t7 rnap,pet- 32a-iers-egfp plasmid was transfected into mdbk-t7 cells and the expression of enhanced green fluorescent protein (egfp) was monitored by western blotting. the results showed t7 rnap has been stably integrated into mdbk-t7 cells and has complete transcription activity,which provides an efficient tool for bvdv rescue. copyright © 2014 zoological society of pakistan.