摘要目的 将丙型肝炎病毒(Hepatitis C virus,HCV)包膜糖蛋白E_2与脂联素球部结构域(gAD)进行基因融合后构建DNA疫苗,并通过免疫小鼠评价其所诱发的HCV E_2特异性细胞免疫应答.方法 根据E_2与gAD的基因序列设计引物,分别扩增出不含跨膜区的E_2基因和添加连接肽的gAD基因,再利用重叠PCR将二者融合,以pVRC为载体构建DNA疫苗质粒,经体外转染293T细胞并用Western Blot证实其表达后,采用皮内注射加卡钳电极电转的方式于0、3、6周免疫BALB/c小鼠,取脾细胞进行ELISPOT检测分析其所诱发的E_2特异性T细胞免疫应答水平.结果 成功构建了能有效表达E_2-gAD融合蛋白的新型DNA疫苗,经皮内电转方式免疫小鼠后可获得比单独E_2疫苗更强的HCV特异性T细胞免疫应答.结论 gAD基因的融入可在一定程度上增强疫苗的免疫原性,为新型HCV DNA疫苗的进一步发展和优化莫定基础.

abstractsObjective To rational design HCV DNA vaccine candidates and evaluate their specific immunity to HCV in mice. Methods We design to construct two DNA vaccine candidates, one consists of E_2 (the envelope glycoprotein 2 of HCV) gene only, the second consists of E_2-gAD (Globular Domain of Human Adiponectin) fusion gene via overlapping PCR. Confirm the expression of the DNA vaccines by Western blotting, and then vaccinated by injection of DNA vaccines with gene electrotransfer (GET) in BALB/c mice. The immune response was measured by IFN-gamma ELISPOT. Results The DNA vaccine candidate consists of E_2-gAD could effectively express in vitro , and it could induced a higher anti-HCV T cell response in mice than the one consists of E_2 only. Conclusion The HCV DNA vaccine consists of E_2-gAD fusion can increase the immunity of the E_2 to some extend, and the research paved a way to develop and optimize the novel HCV DNA vaccine.