RESUMO
Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.
RESUMO
Objective To construct a hexon-chimeric human adenovirus type 3 ( HAd3 ) vector expressing two neutralizing epitopes of hepatitis B surface antigen preS 1 (HBsAg-preS1) and to analyze the antigenicity of the chimeric epitopes .Methods Two neutralizing epitopes of HBsAg-preS1 including KR359 and KR127 were inserted into hypervariable region 1 ( HVR1) and hypervariable region 2 ( HVR2) of HAd3 hexon .Chimeric hexon gene encoding the two epitopes was amplified by overlap PCR and then subcloned in -to shuttle plasmid pBR322-L/R containing the homologous recombination regions .The digested shuttle plas-mid containing chimeric hexon gene was co-transfected into Escherichia coli BJ5183 cells together with back-bone plasmid pBRAdΔE3GFP to construct pBRAdΔE3GFP-preS1 vector.Then pBRAdΔE3GFP-preS1 vector was digested with AsiSⅠand transfected into AD293 cells to construct recombinant virus (rAD3E-preS1). CsCl gradient centrifugation was used for purification .Glutathione S-transferase ( GST ) fusion protein KR359KR127 ( GST-KR359KR127 ) was expressed in Escherichia coli BL21 by using expression vector pGEX-4T3.Female BALB/c mice at age 6-8 weeks were intraperitoneally injected with 1010 virus particles or 80 μg GST fusion protein .Serum samples were collected to analyze the antigenicity of two epitopes by ELISA and Western blot .Results ELISA showed that KR 359 and KR127 were successfully exposed on viral sur-faces by using hexon-chimeric HAd3 vector .The induced polyclonal antibodies in serum samples could rec-ognize GST fusion protein and native HBsAg from patients infected with hepatitis B virus .Conclusion The antigen capsid-incorporation strategy could be used to display epitopes on viral surface .Enhanced antigen-specific responses could be achieved through inserting multiple foreign epitopes into hexon HVRs .This study provided evidence for further application of hexon -chimeric human adenovirus type 3 vector in the developmentof vaccine, especially for the development of multivalent hepatitis B vaccine .