为构建流行性乙型脑炎病毒(JEV)E蛋白多表位疫苗,本研究将人工合成的优化后的E蛋白多表位肽(MP)序列克隆入酵母表达载体pPICZα-A,构建分泌型重组酵母表达质粒pPICZ-MP,经Sac Ⅰ酶切线性化后电转化导入毕赤酵母X-33,阳性重组子经甲醇诱导表达,亲和层析纯化重组蛋白(rMP).通过测定rMP免疫小鼠的抗体水平、抗体亚型、中和抗体和CTL,以评价该多表位疫苗在小鼠模型上的免疫原性.结果表明:成功构建了表达JEV E蛋白多表位抗原的重组酵母菌株X33 (pPICZ-MP),能分泌表达多表位肽rMP.纯化后的rMP免疫小鼠产生了较强的体液免疫和细胞免疫反应,其中和抗体水平、CTL活性均与活病毒疫苗组差异不显著(p＞0.05),且能保护小鼠免受致死量JEV的攻击.构建的JEV E蛋白多表位疫苗具有良好的免疫原性,能够刺激小鼠产生特异性的细胞免疫和体液免疫应答反应,为流行性乙型脑炎多表位疫苗研制提供新的思路.%This study aimed to construct a multi-epitope vaccine of Japanese encephalitis virus (JEV) envelope protein and evaluate its immunogenicity and protective efficacy in mice model.In this study,A codon optimized multiple-epitope peptide gene (MP) from E protein of JEV was cloned into the plasmid pPICZα-A to construct the recombinant expression vector pPICZ-MP.The resulting plasmid linearized with Sac Ⅰ was transformed into Pichiapastoris X-33 by electroporation.The positive clones were selected by Zeocin plates and confirmed by PCR.The supernatant from the shake flask culture was collected and purified with Ni-NTP column.The mice immunized with the recombinant multi-epitope peptide (rMP),and the level of antibody titers,antibody isotypes,neutralizing antibody and CTL were detection to evaluate the immunogenicity and protective efficacy in mice model.The results showed that rMP was highly immunogenic and was able to elicit high titer neutralizing antibodies and cell-mediated immune responses.Our findings indicated that the multi-epitope vaccine rMP might be an attractive candidate vaccine for the prevention of JEV infection.A multi-epitope vaccine of JEV was constructed successfully.Animal experiments showed that immunization with rMP was able to induced high humoral and cellular immune responses,and protect against lethal JEV challenge in mice.The neutralizing antibody level and CTL of mice immunized with the protein showed no significant difference with live JEV vaccine (p＞0.05).The results indicated that the rMP could induce strong immune responses,suggesting that it might be an attractive candidate vaccine for preventing JEV infection.
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