Studies On The Constuction And Expression Of Baculovirus Display System

Based on site-specific transporation of an expression cassette into a baculovirus shuttle vector(Bacmid)which propagated in Escherichia coli,the Bac to Bac System provides a rapid and efficient method to generate recombinant vectors and is widely used for high level expression of heterologous proteins from viruses,fungi,plant,and animals.In this study,we use ph-ph,Mcherry and IRES genes to constuct three shuttle vectors to infact Sf-21 cells.In the process,we should watch ODV and different colours under fluorescence microscope that further enrich the Bac to Bac System.This modified system was also used to express SLAM,the receptor of measles virus,by Dr.Xin Liu in Wuhan College.With the egfp as marker,she found that SLAM was expressed on the surface of Sf-9 cells and it could mediate measles virus infection into the nonpermissive insect cells,indicating that this egfp-harboring system facilitate baculovirus-based local gene function investigation using confocal microscopic technology.In order to study the expression of ph-ph,Mcherry and IRES genes,three recombinant bacmids,pFBDM-ph-ph,pFBDM-Mcherry and pFastBacl-IE2-red-IRES-egfp were constructed by transposing heterologous ph-ph gene driven by AcMNPV polyhedrin promoter and intact Mcherry and IRES gene into the polyhedrin locus of AcBacmid and AeBacmid-egfp through two Bac to Bac system respectively.Three recombinant vectors,pFBDM-ph-ph,pFBDM-Mcherry and pFastBacl-IE2 -red-IRES-egfp were obtained by transfecting three recombinant bacmids into insect cells respectively.4-5 days later,the polyhedrin-egfp fusion expression cassette was transposed into bacmid genome at the attTn7 locus to generate the recombinant vector Bac-ph/pol,which expressed the ph/EGFP fusion protein in insect Sf-21cells.4 days after Bae-gfp/pol DNA transfected into Sf-21cells, occlusion bodies formation and EGFP expression was observed under phase-contrast or fluorescence microscope,and fluorescence was only located on the surface of occlusion bodies,no fluorescence appearing in cytoplasm and other part of nucloplasm. It indicated that EGFP was displayed on the surface of occlusion bodies through fusion with polyhedrin.But the recombinant virus vAc-ph/gfp yielded than occlusion bodies than pFBDMoph in Sf-21cells,and the recombinant occlusion bodies were irregular in morphogenesis observed under electron microscope.Meanwhile pFBDM-Mcherry-Acbacmid-egfp and pFastBacl-IE2-red-IRES-egfp- Acbacmid which have red and green colours with different light to deal with infacted cells,it confirmed that the Mcherry and IRES are expressed in cells.On the other hand, pFBDM-Mcherry-Acbacmid-egfp and pFBDM-ph-ph were extracted and put into Sf-21 cells as second infaction.The results showed that heterologous Mcherry protein was assembled onto the envelope of recombinant ODVs and ODVs also showed red colours.In the last,we used the homologous reorganization to construct a kind of cell wall synthesis flaw E.coli DH10B-asd~-,asd gene can code two aminos pimelic acid (DAP),is the cell wall important composition.As a result of asd gene flaw,without the DAP,the bacteria can not synthesize the cell wall.The pGB2-inv vector contains the inv gene,could express invading protein.We transform the pGB2-inv vector into E.coli DH10B-asd,and then,electroporate pFBDM-ph-ph-AcBacmid-gfp into E.coli DH10B-asd-PGB2-inv.constructs strain DH10B-asd~- inv -PFBDM- ph-ph-Acbacmid -egfp.We directly mix E.coli DH10B-asd~--pGB2-inv with insect cell sf21.Without the DAP,the bacteria cannot synthesize the cell wall,and lysis.AcBacmid-gfp releases, replicates,,assembly,and expresses gfp(green fluorescence protein).and ODV.