Construction And Coexpression Of BoIFN-γ And HLyz And Purification Of BoIFN-γ

1 Construct and coexpression of BoIFN-γand hLyzBoIFN-γgene from pFAST-IFN vector and IRES gene from pIRES vector were respectively subcloned into pFASTBAC-hLyz vector. The recombinant transfer vector was named as pFASTBAC-IFN-IRES-hLyz. Then the vector was transformed into the competent cells of E.coli DH10Bac which contain the bacmid with a mini-attTn7 target site and the helper plasmid. Recombinant bacmid-IFN-IRES-hLyz was generated by transposing the mini-Tn7 element located in pFASTBAC-IFN-IRES-hLyz to the mini-attTn7 attachment site on the bacmid. The result of PCR showed BoIFN-γgene and hLyz gene was successfully inserted into the bacimid. Subsequently the recombinant Bacmid-IFN-IRES-hLyz was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus. The results showed that both BoIFN-γand hLyz were expressed in Sf9 Cells.2 Purification of BoIFN-γexpressed in insect cellsSf9 insect cells wereinfected with the recombinant baculovirus rBac-BoIFN-γat different multiplicity of infection. The cells supernatant were collected at different time to measure the activity of BoIFN-γexpression product. Optimal factors were selected. Pure BoIFN-γwas obtained from the pouch of Sf9 cells infected with the rBac-BoIFN-γby chromatograph.