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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。
Objective To construct a Hepatitis C gene vaccine containing a novel adeno-associated virus (AAV) as a carrier and an E2 antigen of type 1b hepatitis C virus (HCV) envelope protein, and lay a foundation for further research. Methods Serum RNA was extracted from type 1b patients with chronic hepatitis C and the HCV E2 fragment was amplified by RT-PCR. The recombinant plasmid pAAV.CMV.HCV.E2 was constructed and transfected into HEK293 cells. The recombinant plasmid was screened for expression of AAV vaccine Packaging and purification. Results After digestion and sequencing, the AAV plasmid pAAV.CMV.HCV.E2 containing HCV antigen of interest was successfully constructed and transfected into HEK293 cells efficiently to express the antigen of interest. Hepatitis C virus vaccine rAAV2 / 8.CMV.HCV.E2, rAAV2 / rh32.33.CMV.HCV.E2 was successfully packaged and transfected into HepG2 cells efficiently to express the antigen of interest. Conclusion The HCV-E2 vaccine with a novel AAV vector was successfully constructed.