The Establishment Of A High-effective Select Strategy For Recombinant Baculoviruses

Bacuiovirus expression vector system(BEVS) has been developed into a kind of popular expression system for the eukaryotic protein and applied in the regulation of gene expression,the analysis of the structure and function of target proteins,the production of bioactive substance,gene therapy and so on. However,one major constraint for BEVS is the purification of recombinant baculovirus.The traditional strategy is to get recombinant baculovirus by plaque purification,which is low-effective but high-technical.Therefore,a novel strategy for selecting the recombinant viruses is demonstrated in this article:a tag is displayed on the surface of recombinant viruses which is based on the baculovirus surface display system.So that the recombinant viruses are purified by affinity chromatography using immobilized glutathione.A transfer vector,gst-gp64-rfp-pFastBacl,was constructed in this paper and recombinant bacmid,gst-gp64-rfp-Bacmid,was obtained by means of Bac-to-Bac.Red fluorescence was observed in the infected sf9 cells 48h after being transfected by gst-gp64-rfp-Bacmid,which indicates that rfp gene is expressed in sf9 cells.The transcription of gst-gp64 gene is detected by RT-PCR.Two recombinant bacmids,gst-rfp-Bacmid,gp64-rfp-Bacmid,are also constructed as control and expression of exogenous genes,rfp,gst,gp64,are also detected in the infected sf9 cells.Three kinds of virus dilutions are applied to the affinity chromatography using immobilized glutathione respectively.Red fluorescence did not be observed in the sf9 cells which are infected by the eluent of gp64-rfp-Bacmid or gst-rfp-Bacmid.On the opposite,red fluorescence can be observed in the sf9 cells which are infected by the eluent of gst-gp64-rfp-Bacmid.In conclusion, only the recombinant virus displaying the GST-gp64 fusion proteins can be captured by the affinity medium,and eluted using reduced glutathione. The above results proved that it is a convenient method to purify the recombinant viruses from the mixture by affinity medium,and it also raises the efficiency of constructing the BEV.