EV71病毒中和表位和诺如病毒P结构域嵌合蛋白的原核表达

发布时间：2019-02-17 21:13

【摘要】：目的:构建肠道病毒71型(Enterovirus71,EV71)的线性中和抗原表位与诺如病毒P结构域融合基因的重组质粒,在大肠杆菌中表达诺如病毒P结构域与EV71中和抗原表位的嵌合蛋白。方法:根据已报道的3个EV71线性中和抗原表位的氨基酸序列,按大肠杆菌密码子表达使用的偏好性优化和设计各线性中和抗原表位的核苷酸序列,将这些表位以单个或不同的组合克隆至含诺如病毒P结构域和GST标签的质粒中,经测序确认后,分别转化到E.coli BL21(DE3)感受态细胞中,通过IPTG诱导融合蛋白表达。用GST融合蛋白纯化磁珠对融合蛋白进行纯化,最后通过免疫印迹法确认融合蛋白的表达及嵌合蛋白的抗原性。结果:测序结果表明,成功地构建了含EV71病毒3个单表位和4个串联中和抗原表位的诺如病毒P结构域重组质粒,而且这7个含线性中和抗原表位的嵌合蛋白在大肠杆菌中都以可溶形式得到了表达。免疫印迹分析表达蛋白的抗原性结果表明,表达的嵌合蛋白都能与抗诺如病毒P结构域抗血清反应。除了含单表位的SP55和SP28嵌合蛋白外,其它的嵌合蛋白均能与抗EV71病毒的抗血清反应。结论:成功地在大肠杆菌中表达了诺如病毒P结构域和EV71病毒中和抗原表位的嵌合蛋白,且具有抗原性,这为诺如病毒和EV71病毒的二价疫苗及检测方法的研发奠定了基础。
[Abstract]:Aim: to construct the recombinant plasmid of the fusion gene of the linear neutralizing antigen epitope of enterovirus 71 (Enterovirus71,EV71) and the P domain of norovirus, and to express the chimeric protein of the P domain of norovirus and EV71 neutralizing antigen epitope in Escherichia coli. Methods: according to the reported amino acid sequences of three EV71 linear neutralizing antigen epitopes, the nucleotide sequences of each linear neutralizing antigen epitope were optimized and designed according to the preference of E. coli codon expression. These epitopes were cloned into plasmids containing norovirus P domain and GST tag in a single or different combination. After sequencing, these epitopes were transformed into E.coli BL21 (DE3) competent cells, and the fusion protein expression was induced by IPTG. The fusion protein was purified by GST fusion protein and the expression of fusion protein and the antigenicity of chimeric protein were confirmed by Western blotting. Results: sequencing results showed that the recombinant plasmids containing 3 single epitopes and 4 tandem neutralizing antigen epitopes of EV71 virus were successfully constructed. Moreover, the seven chimeric proteins containing linear neutralizing antigen epitopes were all expressed in soluble form in Escherichia coli. The results of immunoblotting analysis showed that the expressed chimeric proteins could react with anti-norovirus P domain antiserum. Except SP55 and SP28 chimeric proteins containing monoepitopes, the other chimeric proteins can react with antiserum against EV71 virus. Conclusion: the chimeric proteins of P domain and neutralizing epitope of EV71 virus were successfully expressed in Escherichia coli, which laid a foundation for the development of bivalent vaccine and detection method for Norovirus and EV71 virus.
【作者单位】：中国医学科学院&北京协和医学院医学生物学研究所感染和免疫实验室;
【基金】：国家自然科学基金(81571549) 云南省重点新产品开发专项项目(2016BC004) 中国医学科学院医学与健康科技创新工程协同创新团队项目(2016-12M-3-026)资助项目
【分类号】：R373

【相似文献】