【摘要】 目的构建以新型腺相关病毒(AAV)为载体,包含1b型丙型肝炎病毒(HCV)包膜蛋白E2目的抗原的丙型肝炎基因疫苗,为进一步研究奠定基础。方法提取慢性丙型肝炎1b型患者血清RNA,用RT-PCR的方法扩增HCV E2片段,构建pAAV.CMV.HCV.E2重组质粒,转染HEK293细胞,筛选出高效表达的重组质粒进行AAV疫苗的包装和纯化。结果经酶切及测序证实,包含HCV目的抗原E2的AAV质粒pAAV.CMV.HCV.E2构建成功,并可高效转染HEK293细胞,表达目的抗原。丙型肝炎基因疫苗r AAV2/8.CMV.HCV.E2,r AAV2/rh32.33.CMV.HCV.E2包装成功,并可高效转染HepG2细胞,表达目的抗原。结论成功构建了以新型AAV为载体的HCV-E2疫苗。更多还原

【Abstract】 Objective To construct novel AAV-associated genetic vaccines for HCV,and lay the foundation for further research. Methods The serum of a genotype 1b hepatitis C patient was collected,and full-length sequence of the envelope protein of hepatitis C virus type 1b was amplified by RT-PCR. Then the target segments were cloned into the AAV cisplasmid,and the recombinant AAV plasmids transfect into HEK293 cells. Recombinant AAV plasmid that could express high levels of antigens were selected for packing and purifying of AAV vaccine. Results After confirmation by restriction endonuclease digestion and sequencing,recombinant AAV plasmids encoding HCV E2 protein were identified. The recombinant AAV plasmids were able to transfect HEK293 cells with high efficacy,and the results of Western blot indicated that HCV E2 protein was high-expressed in HEK 293 cells. Novel AAV-associated genetic HCV vaccines were packaged successfully,and they were able to transfect HEK 293 cells to express high level of HCV E2 protein. Conclusion Novel AAV-associated genetic vaccines for HCV were constructed successfully.更多还原