【摘要】 目的　构建柯萨奇 B1病毒 (CVB1)的 DNA疫苗 ,评价其免疫学性状。方法　将重组质粒 p MD18-T- VPl中的 CVB1VP1基因亚克隆至 p CEP4载体 ,构建真核表达系统 p CEP4 - VP1作为 DNA疫苗 ,经酶切、PCR和测序鉴定后转染至 He L a细胞 ,用间接免疫荧光法检测 CVB1抗原的体外表达。提纯制备 p CEP4 - VP1接种 BAL B/ C小鼠 ,取血清用 EL ISA法检测抗 CVB1Ig M和 Ig G,用 51 Cr释放法测定 CTL反应。结果　酶切、PCR和测序证明 VP1DNA疫苗构建正确 ;转染 p CEP4 - VP1的 He L a细胞中可见到荧光标记 ,提示表达了 VP1蛋白 ;接种 p CEP4 - VP1的小鼠可检测到抗 CVB1的 Ig M和 Ig G,并能产生较强的特异性 CTL反应。结论　 p CEP4 -VP1可作为 DNA疫苗诱导小鼠产生体液免疫和细胞免疫应答更多还原

【Abstract】 Objective To construct DNA vaccine of Coxsackievirus B1(CVB1) and evaluate its immunological characteristics.Methods pCEP4 VP1 was constructed by subcloning the VP1 gene from pMD18 T VP1 into pCEP4 and confirmed with restriction analysis, PCR and DNA sequencing. As a candidate DNA vaccine, pCEP4 VP1 was transfected into HeLa cells for immunofluorescence test. BALB/C mice were inoculated with pCEP4 VP1 and sera harvested from mice were applied for ELISA to detect IgM and IgG. 51 Cr release method was used to investigate specific CTL.Results Restriction analysis, PCR and DNA sequencing showed that pCEP4 VP1 was constructed correctly. Fluorescence signals can be seen on HeLa cells transfected with pCEP4 VP1, which suggest that CVB1 VP1 antigen could be expressed in vitro. The inoculated mice could produce CVB1 specific IgM,IgG and CTL response.Conclusions pCEP4 VP1 may play as a DNA vaccine to induce humoral and cytologic immune response in mice.更多还原