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目的原核表达埃可病毒6型表面特异性蛋白VP1,并制备其多克隆抗体。方法筛选VP1蛋白氨基酸序列中抗原性强的片段,优化基因序列,分别构建重组表达质粒p GEX-4T-2-vp1和p ET-28a-vp1。将经双酶切及测序鉴定正确的重组质粒转化E.coli BL21(DE3),IPTG诱导表达,进行15%SDS-PAGE鉴定。将融合蛋白VP1-GST及VP1-His分别用Glutathione resin和High Affinity Ni-NTA resin进行纯化。以纯化后的VP1-His融合蛋白作为免疫原免疫新西兰大耳白兔,制备多克隆抗体,以VP1-GST融合蛋白为检测抗原,间接ELISA法检测血清抗体效价,Western blot法检测血清抗体特异性。结果经双酶切及测序鉴定证明,重组质粒p GEX-4T-2-vp1和p ET-28a-vp1构建正确。VP1-GST和VP1-His融合蛋白的相对分子质量分别为38 000和19 000,两种融合蛋白分别以可溶性蛋白和包涵体形式表达,约占菌体总蛋白的60%和30%,纯化后的纯度均>90%。兔抗VP1血清效价为1∶320 000,可与融合蛋白VP1-GST和VP1-His发生特异性结合。结论成功原核表达了融合蛋白VP1-His和VP1-GST,且VP1-His具有良好的免疫原性,其制备的多克隆抗体的特异性较好,为埃可病毒快速检测技术的建立奠定了基础。
Objective To express eukaryotic expression vector VP1 of echovirus type 6 and prepare its polyclonal antibody. Methods The strong antigenic fragment in the amino acid sequence of VP1 protein was screened and the gene sequence was optimized. The recombinant expression plasmids pGEX-4T-2-vp1 and p ET-28a-vp1 were constructed respectively. The recombinant plasmid was transformed into E.coli BL21 (DE3) by double enzyme digestion and sequencing. The expression was induced by IPTG and identified by 15% SDS-PAGE. The fusion proteins VP1-GST and VP1-His were purified using Glutathione resin and High Affinity Ni-NTA resin, respectively. The purified VP1-His fusion protein was used as immunogen to immunize New Zealand white rabbits to prepare polyclonal antibody. The VP1-GST fusion protein was used as the detection antigen, the indirect ELISA was used to detect the serum antibody titer, and the serum antibody was detected by Western blot Sex. Results Double enzyme digestion and sequencing proved that the recombinant plasmids pGEX-4T-2-vp1 and p ET-28a-vp1 were constructed correctly. The relative molecular masses of VP1-GST and VP1-His fusion proteins were 38 000 and 19 000, respectively. The two fusion proteins were expressed as soluble proteins and inclusion bodies respectively, accounting for 60% and 30% of the total bacterial proteins. After purification Purity> 90%. Rabbit anti-VP1 serum titer of 1: 320 000, with the fusion protein VP1-GST and VP1-His specific binding occurs. Conclusion The fusion proteins VP1-His and VP1-GST were successfully expressed in prokaryotic cells, and VP1-His was immunogenic. The specificity of the prepared polyclonal antibodies was good, which laid the foundation for the rapid detection of echovirus .