埃可病毒6型表面特异性抗原VP1的原核表达及其多克隆抗体的制备

Prokaryotic expression of surface specific antigen of echovirus 6and preparation of its polyclonal antibodies

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 the surface specific antigen VP1 of echovirus 6 in prokaryotic cells and prepare its polyclonal antibodies. Methods The fragments with strong antigenicity was screened from the amino acid sequence of VP1 protein, of which the sequences were optimized and inserted into expression vectors p GEX-4T-2 and p ET-28a（＋）respectively. The constructed recombinant plasmids p GEX-4T-2-vp1 and p ET-28a-vp1 were identified by restriction analysis and sequencing, then transformed to E. coli BL21（DE3）for expression under induction of IPTG. The expressed product was identified by SDS-PAGE. Fusion proteins VP1-GST and VP1-His were purified by Glutathione resin and High Affinity Ni-NTA resin chromatography respectively. New Zealand rabbits were immunized with the purified VP1-His, and the prepared polyclonal antibody was determined for serum antibody titer by indirect ELISA using VP1-GST as a detection antigen, and tested for specificity by Western blot. Results Restriction analysis and sequencing proved that the recombinant plasmids p GEX-4T-2-vp1 and p ET-28a-vp1 were constructed correctly. Fusion proteins VP1-GST and VP1-His, with relative molecular masses of about 38 000 and about 19 000, were expressed in a soluble form and in a form of inclusion body, contained about 60% and about 30% of total somatic proteins, respectively, both of which reached purities of more than 90% after purification. The rabbit antiserum against VP1 reached a titer of 1 ∶ 320 000, and showed specific binding to VP1-GST and VP1-His fusion proteins. Conclusion Fusion proteins VP1-His and VP1-GST were successfully expressed in prokaryotic cells, which showed high immunogenicity, and the prepared polyclonal antibodies showed high specificity. It laid a foundation of development of detection method for echovirus.