title: [Secretion expression in Pichia pastoris and immunogenicity comparison of two forms of HBsAg/GM-CSF fusion proteins].
abstract: OBJECTIVE
  To express two fusion forms of hepatitis B virus surface antigen (HBsAg-s and s-HBsAg) in the Pichia pastoris expression system, and compare immunogenicity of the two fusion proteins.
  
  
  METHODS
  Was fused GM-CSF to 5' or 3' terminal of HBsAg by inserting the gene fragment of connecting peptide (Gly(4)Ser)(3) to linker gene of GM-CSF and HBsAg. The two fusion proteins were expressed by secreting type expression plasmid pPIC9K in the Pichia pastoris, then the expressed products were detected by SDS-PAGE, Western blot and purified by DEAE-Sepharose Fast Flow ion exchange columns. Mice were inoculated with the two purified HBsAg/GM-CSF fusion proteins and HBsAg respectively in each, and the levels of anti-HBsAg in mice sera were tested by ELISA.
  
  
  RESULTS
  Two HBsAg/GM-CSF fusion proteins were successfully expressed in the form of secretion in Pichia pastoris strain GS115, and exhibited specific reaction with both anti-HBsAg and anti-GM-CSF antibodies in Western blot. ELISA results showed after the inoculation the levels of anti-HBsAg induced by the two HBsAg/GM-CSF fusion proteins was higher than by HBsAg alone (P<0.05). Furthermore, the effect by fusing GM-CSF to C terminal of HBsAg was better than by fusing GM-CSF to N terminal of HBsAg.
  
  
  CONCLUSIONS
  The immunogenicity of HBsAg could be enhanced by fusing GM-CSF.
authors.affiliation: Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences and Union Medical University, Kunming, China. yi_shan@126.com
authors.forename: Shan
authors.initials: S
authors.lastname: Yi
authors.forename: Xiao-Rong
authors.initials: XR
authors.lastname: Xi
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Ding-Feng
authors.initials: DF
authors.lastname: Li
authors.forename: Mao-Sheng
authors.initials: MS
authors.lastname: Sun
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D005260:Female; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000906:Antibodies; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor