Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells

Abstract:

Abstract:［Objective］ To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)，to highly express Newcastle disease virus(NDV) F gene in the primary chicken embryo cells． ［Method］We extracted total RNAs from NDV La Sota strain． Then the F gene was amplified by reverse transcription polymerase chain reaction． We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3＇ end，which expressed under the control of the CMV promoter．The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus．After amplification of recombinant baculovirus，the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection，and the proteins were harvested at 72 h after infection．The F protein expression levels mediated by WPRE regulatory element were analyzed．［Results］ Western blot results show that the F gene was successfully expressed in chicken primary cells． The product was a 56kDa protein and could be recognized by anti-NDV serum．The WPRE fusion significantly improved the F gene expression as 10mmol/L butyrate did，but different to butyrate，the WPRE regulatory element was nontoxic to cells．［Conclusion］The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein．In addition，the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells．The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.