title: [Construction of recombinant adeno-associated virus carrying hepatitis B surface antigen gene and preliminary study of the gene expression and function].
abstract: OBJECTIVE
  To construct recombinant adeno-associated virus (rAAV) carrying hepatitis B surface antigen (HBsAg) gene and study the function of the expressed HBsAg.
  
  
  METHODS
  HBsAg gene (subtype ayw) was amplified from PTHBV-1 by PCR and cloned into the adeno-associated virus vector pSNAV to form the recombinant pSNAV-HBsAg, which was transfected into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell line, BHK-HBsAg, was isolated, which was capable of HBsAg expression and was subsequently infected with HSV-1-HSV1-rc/Delta UL2 that was able to package the rAAV. After purification, rAAV-HBsAg was obtained. The expression of HBsAg in BHK-21 cells and 293 cells infected with rAAV-HBsAg were detected by enzyme-linked immunosorbent assay (ELISA), and the HBsAg antibodies in the sera of rAAV-HBsAg-immunized BalB/C mice were assayed by radio immunoassay.
  
  
  RESULTS
  As detected by ELISA, the expressed HBsAg in mixed cell line mounted to 28.6+/-6.72 ng per 5 x 10(6) cells. The BHK-21 cells and 293 cells infected with rAAV-HBsAg were both capable of HBsAg expression, the amount of which augmented with the increase of multiplicity of infection (MOI). BalB/C mice immunized with rAAV-HBsAg produced anti-HBsAg antibodies.
  
  
  CONCLUSIONS
  rAAV-HBsAg can induce humoral immune response against HBsAg and therefore can be a promising candidate hepatitis B vaccine, and in addition, it may serve the purpose of exploring possible immunotherapy for chronic HBV infection.
authors.affiliation: Department of Epidemiology, First Military Medical University, Guangzhou 510515, China. guifanghu@hotmail.com
authors.forename: Gui-fang
authors.initials: GF
authors.lastname: Hu
authors.forename: Xiao-bing
authors.initials: XB
authors.lastname: Wu
authors.forename: Shou-yi
authors.initials: SY
authors.lastname: Yu
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Dong
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.forename: Yun-de
authors.initials: YD
authors.lastname: Hou
mesh_terms: D000818:Animals; D006224:Cricetinae; D000229:Dependovirus / Q000235:genetics*; D006510:Hepatitis B Antibodies / Q000097:blood; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000276:immunology; D017325:Hepatitis B Vaccines / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic