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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。
OBJECTIVE: To construct a recombinant plasmid containing the linear neutralizing epitope of Enterovirus 71 (EV71) fused with norovirus P-domain and express norovirus P domain and EV71 neutralizing antigen in Escherichia coli Bit chimeric protein. METHODS: Based on the reported amino acid sequences of the three linearized neutralizing epitopes of EV71, the nucleotide sequences of each of the linear neutralizing epitopes were optimized and designed according to the preference of E. coli codon usage, Single or different combinations were cloned into the plasmid containing norovirus P-domain and GST tag. After sequencing, they were transformed into E.coli BL21 (DE3) competent cells, and the fusion protein was induced by IPTG. The fusion protein was purified by magnetic beads with GST fusion protein. Finally, the expression of the fusion protein and the antigenicity of the chimeric protein were confirmed by immunoblotting. Results: Sequencing results showed that a recombinant plasmid of pEGV containing 3 single epitopes and 4 tandem neutralization epitopes of EV71 virus was successfully constructed, and these 7 linear neutral epitopes Proteins are expressed in soluble form in E. coli. The antigenicity of the expressed protein by Western blot analysis indicated that the expressed chimeric protein reacted with the anti-Norovirus P domain antiserum, respectively. In addition to the single epitopes of the SP55 and SP28 chimeric proteins, other chimeric proteins were able to react with anti-EV71 antiserum. CONCLUSIONS: Chimeric proteins that successfully expressed the norovirus P domain and the EV71 virus neutralizing epitope in E. coli have been successfully obtained and have antigenicity, which is a bivalent vaccine for Norovirus and EV71 viruses and a test method for R & D laid the foundation.