【摘要】 目的在前期筛选的HSV-2gD模拟抗原表位序列P6的基础上,在GenBank上选取与P6序列相对应且相似的HSV-2天然野生株gD的基因序列NP6,以pcDNA3.1(-)为载体、IL-18为佐剂,构建和表达NP6与IL-18串联的重组表达质粒,并观察免疫小鼠后的效果。方法采用串联简并引物PCR法构建重组质粒pcDNA3.1-IL18-NP6和pcDNA3.1-IL18。将构建的真核表达质粒pcDNA3.1-IL-18和pcDNA3.1-IL-18-HSVNP6肌内注射免疫BALB/c小鼠3次,每次间隔1周。末次免疫后1周眼眶静脉采血,ELISA法检测小鼠血清特异性抗体滴度、IFN-γ及IL-18含量。结果构建的串联重组质粒pcDNA3.1-IL18-NP6和pcDNA3.1-IL18经酶切及测序显示基因序列完全正确,间接免疫荧光、Western blot检测证实模拟抗原表位序列NP6具有近似天然序列的生物学活性;用表达质粒免疫小鼠,可刺激产生高滴度的特异性抗体,并可产生较高水平的IL-18和IFN-γ。结论成功构建和表达了重组质粒pcDNA3.1-IL18-NP6,表达产物具有免疫原性。更多还原

【Abstract】 Objective To construct and express the pcDNA3.1/IL-18-HSV NP6 recombinant DNA vaccine and to study the immune response to this vaccine.Methods NP6 and IL-18 genes were cloned into the eukaryotic expression vector pcDNA3.1(-).The recombinant plasmid pcDNA3.1-IL-18-HSV NP6 was used to inoculate BALB/c mice by intramuscular injection three times every other week.One week after final vaccination,the level of specific IgG antibody,IFN-γ,and IL-18 were detected by ELISA.Results Recombinant pcDNA3.1-IL-18-HSVNP6 plasmids were confirmed by enzyme digestion and DNA sequencing.Indirect immunofluorescence and Western blotting both indicated that the recombinant plasmids were expressed in CHO cells.After inoculation with the pcDNA3.1-IL-18-HSVNP6 vaccine,mice had specific antibodies and higher levels of IFN-γ and IL-18.Conclusion The DNA vaccine pcDNA3.1-IL-18-HSVNP6 was successfully constructed and expressed in eukaryotes.更多还原