人IL-2信号肽基因增强柯萨奇病毒B3型VP1 DNA疫苗诱导的中和抗体应答

Signal peptide DNA of human IL-2 enhanced neutralizing antibody response of Coxsackievirus B3 VP1 DNA vaccine

目的 将人白细胞介素 2 (hIL 2 )信号肽基因与柯萨奇病毒B组 3型 (CVB3)的VP1基因融合 ,构建分泌型VP1真核表达质粒pcDNA3/sVP1;免疫小鼠后 ,通过测定血清特异性中和抗体效价及对致死量CVB3攻击的保护作用 ,评价疫苗的免疫效果。方法 采用重叠区基因扩增法 ,将hIL 2信号肽基因连同其下游 11个氨基酸残基的基因与CVB3VP1基因拼接 ,获得分泌型VP1(sVP1)的基因 ;将sVP1基因克隆至真核表达载体pcDNA3,构建分泌型VP1真核表达质粒pcDNA3/sVP1;肌注免疫小鼠 ,测定血清中CVB3特异性中和抗体的效价 ;第 3次免疫后 2周 ,腹腔内注射 10 0 0TCID50 的CVB3,观察小鼠生存情况。结果 成功构建了分泌型VP1真核表达质粒pcDNA3/sVP1,插入的sVP1基因中含有hIL 2信号肽及其下游 11个氨基酸残基的基因以及VP1基因 ;pcDNA3/sVP1可比对照质粒pcDNA3/VP1诱导小鼠产生更高水平的中和抗体。结论 hIL 2信号肽基因增强了柯萨奇病毒B3型VP1DNA疫苗诱导的中和抗体应答 。

Objective To construct a secretable eukaryotic expressing plasmid pcDNA3/sVP1. The immune effects were evaluated by determining the specific neutralizing antibody titration and observing the survival of the mice after challenge with a lethal dose of the virus. Methods The signal peptide DNA sequence of hIL 2 was fused to 5′ terminal of VP1 gene by overlap extension and the sVP1 was cloned into eukaryotic expressing vector pcDNA3 to construct pcDNA3/sVP1. The inserted gene fragment was identified by sequencing and restriction enzyme analysis. BALB/c mice were inoculated with plasmids intramuscularly (i.m.) at 2 week intervals. Thirteen days after every injection, sera were collected and the titer of CVB3 specific neutralizing antibodies was measured. Two weeks after the third immunization, mice were subjected to intraperitoneal (i.p.) challenge with 1000 TCID 50 CVB3 and the numbers of surviving animals were monitored up to 3 weeks post infection. Results The secretable eukaryotic expressing plasmid pcDNA3/sVP1 was successfully constructed. The mean neutralizing antibody titer after the third immunization induced by pcDNA3/sVP1 was much higher than that induced by pcDNA3/VP1( P < 0.001 ), showing that pcDNA3/sVP1 could elicit significantly higher level of neutralizing antibody response as compared with pcDNA3/VP1. Conclusion Fusing the signal peptide sequence of hIL 2 to VP1 gene enhanced the neutralizing antibody response of the DNA vaccine.