title: Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles.
abstract: OBJECTIVE
  To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).
  
  
  METHODS
  The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.
  
  
  RESULTS
  The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.
  
  
  CONCLUSIONS
  The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118.
authors.forename: Hong-Zhao
authors.initials: HZ
authors.lastname: Li
authors.forename: Hong-Ying
authors.initials: HY
authors.lastname: Gang
authors.forename: Qiang-Ming
authors.initials: QM
authors.lastname: Sun
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.forename: Yan-Bing
authors.initials: YB
authors.lastname: Ma
authors.forename: Mao-Sheng
authors.initials: MS
authors.lastname: Sun
authors.forename: Chang-Bai
authors.initials: CB
authors.lastname: Dai
mesh_terms: D000939:Epitopes; D005818:Genetic Engineering; D018963:Hepatitis Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology*; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014614:Vaccines, Synthetic
chemical_list: D000939:Epitopes; D018963:Hepatitis Antigens; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic