| HCV infection has been one of the most important damaging infectious diseases world-wide. Without a reliable cell culture system in vitro and successful infection model, study on HCV and prevention of hepatitis C have been extremely limited. Construction and packing of replication-defective adenovirus vectors expressing HCV structural proteins will play an important role in research of HCV and vaccination.HCV infection often result in persistent infection. Moreover, it has been known that chronic HCV infection is associated with high risk of liver cirrhosis and hepatocellular carcinoma but without perfect prevention and therapeutic procedure. Exploration of new way to vaccination and clearance of HCV will be a great challenge. T-cell vaccine is being noticed as a new way of HCV prevention and clearance, and research on preparation of T-cell vaccine and its clearance against HCV will contribute to it.We constructed 3 kinds of shuttle plasmid encoding HCV structural proteins by means of molecular cloning technique; based on it, 3 kinds ofadenovirus vectors of HCV structural proteins were packed, screened and identified. We also selected and synthesized 5 kinds of CTL epitope peptides of HCV structural gene which induced 5 kinds of corresponding HCV specific CTL, which were evaluated by experiments in vivo and in vitro. Furthermore, we discussed the influence of immune-sensibilization before induction to the activity of T-cell vaccine, compared the immune-sensibilization difference between HCV-adenovirus vectors and plasmid vectors. We also compared T-cell vaccine with DNA vaccine on in vivo inhibition to HCV transplanting tumor to evaluate the difference effects of protection from HCV infection. Conclusion:1 The 3 constructed adenovirus vectors shuttle plasmid of HCV structural gene. pAd.HCV-C. pAd.HCV-CEl and pAd.HCV-CE!E2. were confirmed to be inserted the sequences of HCV C, C+E1 and C+E1+E2 by double endonucleases, PCR and sequencing.2 After co-transfection with the 3 kinds of shuttle plasmid and adenovirus genome plasmid (p.IM17) into 293 cell. 3 HCV structural protein expressing adenovirus vectors. pAd.HCV-C, pAd.HCV-CEl and pAd.HCV-CE!E2 were packed and screened by virus plaques technique, identified to be good infectivity and expressivity of inserted HCV structural proteins or fusional structural. In contrast to plasmid DNA vector, adenovirus vectors appeared to be better immunizational effects and with the hope of being developed to be vaccine against HCV.3 We also selected and synthesized 5 CTL epitopes peptides of HCV structural gene which were capable of combining with H-2d molecule, established methods of in vitro elicitation and proliferation of specific CTL andobtained 5 kinds of specific CTLs.4 We observed that T cell vaccine activity could be greatly improved through immune-sensibilization to mice with HCV adenovirus vectors or plasmid vector before T-cell vaccine induction, and adenovirus vectors proved to be superior to plasmid vector.5 In vitro specific killing experiments showed that spleen cells induced by PI, P3 and P4, which contained higher proportion of CDg+ cells and generated better CTL activity, could be introduced to in vivo experiment against HCV6 We successfully setup a HCV structural gene in vitro cell-transfection system SP2/0-HCV, through which also setup a murine model with subcutaneous transplanting tumor of HCV. Determined by immunohistochemical technique, there were HCV structural proteins in both SP2/0-HCV and its transplanting tumor tissue.7 When the murine model were immunized by the 5 kinds of elicited HCV-specific CTLs as a role of T-cell vaccine, significant inhibition to HCV transplanting tumor were observed in groups of PL P3. and P4.8 Comparison were performed between the 3 T-cell vaccine and plasmid DNA vaccine expressing HCV structural gene on therapeutic effects in HCV transplanting tumor murine model. The result showed that the former was obviously superior the... |
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