طراحي، ساخت و ارزيابي ايمني زايي سازه هاي DNA پلي توپ با استفاده از اپي توپ هاي غالب ويروس هپاتيت سي در موش BALB/c

Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitop s in ALBIc

Objective: Polytope DNA vaccines , capable of focusing the cytotoxic T lymphocyte (CTL) response on critical epitopes, represent a promising approa ch in HCV immunotherapy. Nevertheless, due to controversial rules governing epitope processing and the low level expression/ immunogenicily of recombinant polytope peptides, design ing and primary expressionl immunogenicity analysis of these vaccine types should be the first consideration prior to costly transgenic animal studies. Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected and designed in three appropriate sequential tandems based on epitope and proteasomal cleavage predictions . The related nucleotide sequences were synthesized using SOEing PCR method and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatiti s B surface antigen (HBsAg-S) gene. Follow ing the preparation of polyclonal anti-sera , expression! secretion of polytopes was evaluated in Coso? cells by using immunofluorescence, Western blot, dot blot, ELISA and RT-PCR techniques . The immunogenicity of the plasmids was also assessed through the delayed -type hypersensitivity (DTH) assay in BALB!c mice. Results: Due to in silico designs and optimizations, the polytope products of constructed plasmids were efficiently detected in vitro through common techniques and HBsAg-S-based particles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmids were able to efficiently indu ce a positive DTH response while HBsAg-S fusion constructs indicated a significant immunopotential effect towards the incorporated mouse epitopes. Conclusion: Designed polytope constructs of this study are efficiently expressed and processed. They have the required initial potency for further immunogenicity analysis in trans qeruc mice.