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目的研究乙型肝炎病毒(HBV)自身增强子Ⅰ(enhancerⅠ,ENHⅠ)对HBVDNA疫苗免疫应答的影响。方法采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原(HBsAg)和HBsAg-ENHⅠ基因片段,重组到载体VR1012中,构建两种HBVDNA疫苗,转染COS-7细胞及HepG2细胞并免疫BALB/c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果转染的HepG2和COS-7细胞均表达HBsAg;连接ENHⅠ的HBVDNA疫苗转染HepG2细胞后HBsAg表达量明显升高,两种疫苗转染COS-7细胞表达HBsAg无明显差异;免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞(CTL),两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHⅠ可使HBVDNA疫苗转染HepG2细胞表达HBsAg明显增加,对转染COS-7细胞表达HBsAg及接种BALB/c小鼠引起的免疫应答无明显影响。
Objective To study the effect of enhancer Ⅰ (ENHⅠ) on the immune response to HBVDNA vaccine in hepatitis B virus (HBV). Methods HBsAg and HBsAg-ENHⅠ gene fragments were amplified by PCR from HBVadr subtype genomic DNA. The recombinant plasmids were subcloned into vector VR1012 to construct two kinds of HBVDNA vaccines and transfected into COS-7 cells and HepG2 cells And BALB / c mice were immunized. Western blotting, ELISA, ELISPOT and other methods were used to detect the expression of COS-7 and HepG2 cells and the humoral and cellular immune response in mice. Results HBsAg was expressed in both HepG2 and COS-7 cells transfected with HBV DNA vaccine; HBsAg expression was significantly increased in HepG2 cells transfected with HBV DNA vaccine of ENHⅠ, and no significant difference was found in the expression of HBsAg between the two vaccines transfected COS-7 cells; HBsAb and HBsAg-specific cytotoxic T lymphocytes (CTLs) were produced in the second week. There was no significant difference between the two vaccines in producing HBsAb and HBsAg-specific CTLs. Conclusions ENHⅠ can significantly increase HBsAg expression in HepG2 cells transfected with HBVDNA vaccine, and has no significant effect on the immune response induced by HBsAg transfection in COS-7 cells and inoculation of BALB / c mice.