【摘要】 目的构建柯萨奇病毒B3(CoxsackievirusB3,CVB3)VP1与白细胞介素 15 (interleukin 15 ,IL 15 )双表达基因疫苗 ,并观察其对小鼠的免疫效果。方法从CVB3VP1基因的重组质粒 pcDNA3/VP1上扩增出带有完整表达构件的VP1基因 ,克隆至IL 15基因重组质粒 pcDNA3/IL 15 ,构建成双表达重组质粒 pcDNA3/VP1 IL 15 ,转化DH5a大肠杆菌 ,大量扩增后 ,肌内注射Balb/c小鼠 ,1次 /周 ,共 3次 ,每次免疫后第 6天取血清 ,用微量中和试验方法检测血清中和抗体。第 3次免疫后的第 7天 ,用 80 0TCID5 0CVB3感染小鼠 ,观察各组小鼠的生存情况。结果构建的 pcDNA3/VP1 IL 15重组质粒目的基因与CVB3 VP1和IL 15的序列相同 ;免疫小鼠后 pcDNA3/VP1 IL 15重组质粒能诱导机体产生中和抗体 ,抗体滴度随免疫次数增加而提高 ;病毒攻击后 pcDNA3/VP1 IL 15和 pcDNA3/VP1+pcDNA3/IL 15组较其他组生存时间明显延长 (P <0 .0 5 )。结论本研究成功构建了 pcDNA3/VP1 IL 15真核双表达重组质粒 ;该质粒能诱导机体产生中和抗体 ,对CVB3毒株的攻击有一定程度的保护作用。更多还原

【Abstract】 ObjectiveTo study the immunological enhancement effect of interleukin-15 (IL-15) as gene adjuvant to Coxsackievirus B3(CVB3) VP1 gene vaccine. MethodsThe whole fragment of CMV-VP1-BGH polyA was amplified by polymerase chain reaction(PCR) from pcDNA3/VP1 and inserted into pcDNA3/IL-15 plasmid. Then pcDNA3/VP1-IL-15 DNA was prepared and purified through transforming into the E.coli DH5a. Balb/c mice were immunized with the DNA vaccine three times at one week interval by tibialis anterio muscle injection. Sera were collected in the sixth day after every immunization for measuring neutralizing antibodies. The mice were infected with 800TCID50 CVB3 after three times of immunization injection and the survival quantities of which were observed after three weeks. ResultsThe sequence of objective gene of eukaryotic expression plasmid pcDNA3/VP1-IL-15 was identical to that of CVB3-VP1 and IL-15, pcDNA3/VP1-IL-15 could induce the production of neutralization antibody and its titers increased with the times of immunization,pcDNA3/VP1-IL-15 and pcDNA3/VP1+pcDNA3/ IL-15 groups survived significantly longer than other four groups after the mice were infected with CVB3.ConclusionpcDNA3/VP1-IL-15 was constructed successfully, it could induce the production of neutralization antibody and could provide protection against the attack of CVB3.更多还原