[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid ...[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.展开更多
基金Supported by National Transgenic Major Program of China(2009ZX08007-006B)the National Natural Science Foundation of China(31072160)+2 种基金Science and Technique Foundation of Shandong Province(2009GG20002032)Natural Science Foundation of Shandong Province(Y2008D20)an Open Issue of State Key Laboratory of Veterinary Biotechnology Fund(SKLVBF200806)~~
文摘[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.
