title: [Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells].
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.
  
  
  METHODS
  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 10 mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells.
  
  
  CONCLUSIONS
  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.
authors.forename: Dongni
authors.initials: D
authors.lastname: Gao
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Ping
authors.forename: Liying
authors.initials: L
authors.lastname: Jin
authors.forename: Wanli
authors.initials: W
authors.lastname: Shen
authors.forename: Gang
authors.initials: G
authors.lastname: Song
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Tang
authors.forename: Qi
authors.initials: Q
authors.lastname: An
authors.forename: Yan
authors.initials: Y
authors.lastname: Shen
authors.forename: Jingping
authors.initials: J
authors.lastname: Ge
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002642:Chick Embryo; D002645:Chickens; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D009521:Newcastle Disease / Q000276:immunology / Q000821:virology*; D009522:Newcastle disease virus / Q000235:genetics* / Q000276:immunology; D011201:Poultry Diseases / Q000276:immunology / Q000821:virology*; D050436:Regulatory Elements, Transcriptional; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D014760:Viral Fusion Proteins; D014765:Viral Vaccines