【摘要】 为研究鸭乙型肝炎病毒核心抗原(DHBcAg)真核表达质粒的免疫原性,分析DHBcAgDNA疫苗诱导的体液免疫应答,首先借助生物信息学方法对鸭乙型肝炎病毒(DHBV)Core基因编码的氨基酸序列进行亲疏水性分析,分别构建DHBcAg全基因(E-DHBc263)及去除疏水性序列的DHBcAg片段(E-DHBc180)的真核表达质粒,间接免疫荧光检测结果显示可在COS7细胞内表达。进一步构建原核表达质粒p-DHBc263和p-DHBc180,仅p-DHBc180可表达蛋白,纯化后作为酶联免疫吸附试验(ELISA)包被抗原,用于DHBcAb的检测。分别用E-DHBc263和E-DHBc180免疫小鼠,采用间接ELISA检测DHBcAb。结果显示,E-DHBc180可诱导免疫小鼠产生DHBcAb免疫应答,加强免疫后效价可达1∶100～1∶400。结果提示,E-DHBc180可作为DHBcAgDNA疫苗,在DHBV感染鸭模型中评价其效果。更多还原

【Abstract】 To investigate the immunogenicity of eukaryotic expression plasmid of duck hepatitis B virus core antigen(DHBcAg)in mice,we first analyzed the hydrophilic and hydrophobic characteristics of the amino acid sequence of DHBcAg by bioinformatics with the Vector NTI Suite software,and constructed the eukaryotic expression plasmids of the complete(E-DHBc263)and partial sequences(only the hydrophilic part,E-DHBc180)of DHBcAg gene.The results showed that both plasmids expressed DHBcAg in transfected COS7 cells detected by indirect immunofluorescence.The prokaryotic expression plasmids,p-DHBc263 and p-DHBc180,were constructed to express DHBcAg for DHBcAb detection,but only p-DHBc180-expressed DHBcAg could be detected by Western blot.Then we immunized BALB/c mice with E-DHBc180 and E-DHBc263.DHBcAb titers in sera from immunized mice were detected by enzyme-linked immunosorbent assay(ELISA)on the plates coated with prokaryotically expressed DHBcAg.The mice primed and boosted with E-DHBc180 produced DHBcAb,and titers were 1∶100-1∶400,while in E-DHBc263-immunized mice DHBcAb level was below the detection level.It suggests that E-DHBc180 could be used as DHBcAg DNA vaccine for further research in its effects on DHBV infection.更多还原