The Expression Of RGDV-P8 And RGDV-Pns10 In Sf9 Insect Cells

Objective: Using the bac-to-bac baclovirus expression system to obtain eukarya product of RGDV-P8 and RGDV-Pns10．Methods: The ORF of RGDV-P8 and RGDV-Pns10 were amplified by PCR from RGDV-P8 and RGDV-Pns10 cDNA, and the fragment was inserted into donor vector pfastbacHTb, The plasmid pfastbacHTb-RGDV-P8 and pfastbacHTb-RGDV-Pns10 were transformed into DH10Bac E.coli cells, and the recombinant shuttle vector Bacmid-RGDV-P8 and RGDV-Pns10 were constructed by the site-specific transposition. Bacmid-RGDV-P8 and Bacmid-RGDV-Pns10 were transfected into Sf9 cells via lipofectamine to generate the recombinant baculovirous . And then the recombinant baculovirous was amplified. When the recombined baculovirous infect sf9 cells again, the protein RGDV-P8 and RGDV-Pns10 were expressed. The expression product was analyzed by SDS-PAGE and verified by western-blot.Results:1．The recombinant donor plasmid pfastbacHT-RGDV-P8 and RGDV-Pns10 were constructed , It was identified by PCR, digestion and sequencing that the RGDV-P8 and RGDV-Pns10 genes were successfully inserted into the vector pfastbacHTb.2．The recombinant shuttle vector Bacmid-cystatin was constructed. It was verified by PCR that the RGDV-P8 and RGDV-Pns10 genes were successfully inserted into the site of the shuttle vector.3．The recombinant RGDV-P8 and RGDV-Pns10 were expressed in bac-to-bac system., SDS-PAGE and Western-blot analysis showed that the molecular weight of fusion RGDV-P8 and RGDV-Pns10 were 48kD and 38 kD. It is similar to the theoretic molecular weight.Conclusion: The recombinant fusion RGDV-P8 and RGDV-Pns10 protein were successfully expressed in Bac-to-Bac baculovirous expression system.