乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的小鼠免疫应答研究

Immune responses in mice induced by bicistronic DNA vaccines expressing hepatitis B virus core antigen and Flt3 ligand gene

目的构建乙型肝炎病毒核心抗原(HBcAg)和Flt3配体(FL)胞外段双表达核酸疫苗,并观察其免疫原性。方法分别将HBcAg、FL基因克隆入pJW4303载体,获得双表达核酸疫苗,体外转染293T细胞检测目的基因的表达。分组免疫BABL／c小鼠,酶联免疫吸附试验(ELISA)检测小鼠血清抗-HBc IgG效价,酶联免疫斑点试验(ELISPOT)检测HBcAg特异性Th1／Th2型细胞因子的分泌水平。结果所构建疫苗在体外均能表达HBcAg和FL,当基因位于内部核糖体切入位点(IRES)元件上游时表达水平明显较优。pJW4303／C／FL免疫组产生的抗-HBc IgG效价和Th1／Th2型细胞因子的分泌水平均显著优于pJW4303／C和pJW4303／FL／C组。结论成功构建双表达核酸疫苗,基因位于上游时表达水平高于下游。FL基因的引入明显增强了HBcAg核酸疫苗的免疫原性。

Objective To construct a bicistronic DNA vaccine encoding hepatitis B virus core antigen （HBcAg） and extracellular fraganent of Fh3 ligand （FL） and to study its immunogenecity in mice. Methods HBcAg and FL genes were cloned into vector pJW4303 ahernatively at either upstream or downstream of the internal ribosome entry site （IRES） element.The expression of HBcAg and FL was examined by transfection of these bicistronic DNA vaccines into 293T cells. Five groups of BABL/c mice were vaccinated with DNA vaccines. Anti-HBc IgG titers and HBcAg specific Th1/Th2 type cytokines secreted by splenocytes were measured by ELISA method and ELISPOT assay,respectively. Results All bicistronic DNA vaccines expressed both HBcAg and FL in vitro and expression level was higher when HBc gene located upstream of IRES element. The triers of anti-HBc IgG and levels of Th1/Th2 type cytokines in mice immunized with pJW4303/C/FL were significantly higher or stronger than that with pJW4303/C and pJW4303/FL/C. Conclusion Bicistronic DNA vaccines encoding HBcAg and FL were constructed. The expression of target antigen gene at upstream of IRES element is clearly better than that at downstream. The inmaunogenicity of HBcAg DNA vaccine is enhanced by the inclusion of FL gene.