Study Of A Recombinant Adenovirus Vector Vaccine Expressing The C Antigen Of HCV

Objective: Hepatitis C is a global health problem that is caused by hepatitis C virus. So far, there is no effective treatment available in clinic. Therefore, HCV vaccine has been urgently needed to prevent and treat this infection.HCV belongs to Flaviviridae. Because the RNA polymerase of HCV lack of reading-proof function, this virus displays a high rate of gene variation that make it escape from the immune impression and is the major difficulties faced in HCV vaccine development by using traditional vaccine development strategy. However, vector-based vaccine, which exogenous target antigen-encoding gene or genes was introduced into bodies by, has been showed to be a promising new way for the development of vaccine. The core antigen sequence of HCV is the most conservation and stable region. It has been reported that the C antigen of HCV could induce an efficacy humoral immune response and cytotoxic T lymphcyte against HCV and may be used as a major target antigen for the development of HCV vector-based vaccine. Compared with plasmid, replication-deficient adenovirus vector system showed more advantages. As the original adenovirus, which it derived from, this virus can infect more types of cell and transfer the target gene more efficiently. Therefore, this vector can express the target gene more efficiently and induce a stronger protective immune response to the target protein that made it a promising vector system for the development of vaccine.In this study, the adenovirus-based recombinant vaccine rAd/C was constructed to explore anew way for the development of HCV vaccine.Method: 1 Amplification and identification of the target gene : The DNA fragment of C was amplified by PCR from the plasmid pEGFP-HCV/C. Gene fragments was inserted into pGEM-T vector. Competent E.coli DH5αwere transformed and selected by Ampicillin, recombinant was identified by electrophoresis analyses and gene sequencing.2 Construction of the eukaryotic expression plasmids: The fragment of C was purified from agarose gel, and cloned into the transfer vector pcDNA3.0 cut by the same endonucleases to construct the transfer plasmids pcDNA3.0/C competent bacterial cells were transformed with the plasmid and selected by Kanamycin, the recombined plasmid extracted was verified by PCR and enzymes cutting.3 Preparation of the recombinant adenoviral transfer plasmid pAdTrack-CMV/C: The gene fragment was purified from agaose gel, and cloned into the transfer vector pAdTrack-CMV cut by the same endonuclease to construct the transfer plasmids pAdTrack-CMV/C. The transformed bacteria was selected by Kanamycin, the recombined plasmid extracted was verified by endonuclease analyses.4 Construction of recombinant adenoviral plasmids: The recombinant plasmid were linearized with Pme I and co-transformed into E. coli strain BJ5183 with pAdEasy-1, the viral DNA plasmid. Recombinant was selected with kanamycin and identified by restriction enzyme analysis. The recombinant adenoviral plasmid was transformed to competent E. coli DH5αfor large preparation and the plasmid obtained was purified by PEG.5 Packaging and amplification of the recombinant adenovirus particle: Linearized by Pac I digestion, the recombinant adenoviral plasmid was transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 according to the manufacturer's guidelines, to produce viral particles,followed by screening the expression of green fluorescent protein(GFP). The expression of green fluorescent protein(GFP),which was monitored as a indictor of successful screening. The supernatant and the cell lysate was collected and the recombinant virus was further amplified in HEK 293 cells for 2 to 4 passages. The passage 4 virus was purified using the adenovirus purification kit.6 Titration of the recombinant adenovirus : Cell-free virus stocks were diluted serially in serum-free medium at ten times, and titrated by plaque forming units (PFU) methods according to Ad-Easy vector system application manual。Results: 1 The clone vector pGEM-T/C was constructed successfully; endonuclease analyses and sequencing result showed that the inserted genes was the same as expected.2 The eukaryotic expression plasmid pcDNA3.0/C was constructed successfully.3 The recombinant adenoviral transfer plasmid AdTrack-CMV/C was generated successfully, endonuclease analyses and sequencing result showed that the inserted genes was the same as expected.4 Recombinant adenoviral plasmid pAd/C was constructed successfully. Restriction digest with Pac I yielded a larger fragment of approximately 30 kb, and a smaller fragment of either 3.0 kb or 4.5 kb.5 Recombinant adenoviruses Ad/C was packaged successfully. The infected cells were lysed by freezen-thawing, and supernatant was used to reinfect fresh HEK 293 cells.6 The titration of recombinant adenovirus Ad/C after passage 4 was as follows: 6.8×108 pfu/ml.Construction: The recombinant adenovirus Ad/C was constructed and packaged successfully, which paved a foundation for the development of the hepatitis C vaccine in future.