[Construction of recombinant adeno-associated virus carrying hepatitis B surface antigen gene and preliminary study of the gene expression and function].
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.
Hu
Gui-fang
GF
Department of Epidemiology, First Military Medical University, Guangzhou 510515, China. guifanghu@hotmail.com
Wu
Xiao-bing
XB
Yu
Shou-yi
SY
Dong
Xiao-yan
XY
Chen
Qing
Q
Hou
Yun-de
YD
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*
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic