[Secretion expression in Pichia pastoris and immunogenicity comparison of two forms of HBsAg/GM-CSF fusion proteins].
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.
Yi
Shan
S
Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences and Union Medical University, Kunming, China. yi_shan@126.com
Xi
Xiao-Rong
XR
Shi
Rui
R
Yang
Fang
F
Li
Min
M
Li
Ding-Feng
DF
Sun
Mao-Sheng
MS
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*
D000906:Antibodies; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor