【作者】 李素梅； 齐永； 徐亭亭； 潘英； 李素芹； 李佳萌； 陈晨； 王新国； 徐艺菲； 李越希；

【Author】 Sumei Li;Yong Qi;Tingting Xu;Ying Pan;Suqin Li;Jiameng Li;Chen Chen;Xinguo Wang;Yifei Xu;Yuexi Li;Huadong Research Institute for Medicine and Biotechniques;School of Basic Medicine,Nanjing Medical University;School of Life Science and Technology,China Pharmaceutical University;

【机构】 南京军区军事医学研究所； 南京医科大学基础医学院； 中国药科大学生命科学与技术学院；

【摘要】 目的在原核细胞中表达埃可病毒6型的表面蛋白VP1,制备其多克隆抗体。方法筛选VP1抗原表位富集区,优化基因序列,分别克隆至质粒pGEX-4T-2和pET-28a(+)中,构建重组表达质粒pGEX-4T-2-vp1和pET-28a-vp1,分别转化大肠杆菌(E.coli)BL21(DE3),经IPTG诱导表达后,分别用High Affinity GST-NTA resin和High Affinity Ni-NTA resin进行纯化。以纯化后的VP1-His融合蛋白为抗原免疫新西兰大白兔,制备多克隆抗体,以VP1-GST融合蛋白为检测抗原,采用间接ELISA法检测血清效价,Western blot法检测特异性。结果经双酶切及测序鉴定,证明质粒pGEX-4T-2-vp1和pET-28a-vp1构建正确;在相对分子质量约39 000和20 000处,分别可见VP1-GST和VP1-His融合蛋白的表达,表达量分别约占菌体总蛋白的25%和30%,发现VP1-GST为可溶性蛋白,而VP1-His主要以包涵体的形式存在,需对VP1-His蛋白进行透析复性,纯度均在90%以上;免疫新西兰大白兔后获得高效价的特异性兔抗血清。结论两种标签的融合蛋白均具有良好的免疫原性和抗原性,所制备的多克隆抗体具有较好的特异性,为埃可病毒检测方法的建立奠定了基础。更多还原

【Abstract】 Objective To express the VP1 of Echovirus 6 in prokaryotic cells and prepare its polyclonal antibody.Methods The extracellular fragment with antigen epitopes of VP1 was selected,of which the gene sequence was optimized and cloned into vectors pGEX-4 T-2 and pET-28 a(+) respectively.The constructed recombinant plasmids pGEX-4 T-2-vp1 and pET-28 a-vp1 were transformed to E.coli BL21(DE3) for expression under induction of IPTG.The fusion proteins VP1-GST and VP1-His were purified by High Affinity GST-NTA resin and High Affinity Ni-NTA resin.Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the purified VP1-His fusion protein,determined for titer by indirect ELISA using VP1-GST fusion protein as detection antigen,and for specificity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmids pGEX-4 T-2-vp1 and pET-28 a-vp1 were constructed correctly.Fusion proteins VP1-GST and VP1-His,with relative molecular masses of about 39 000 and 20 000,contained about 25% and 30% of total somatic proteins,respectively.The fusion protein VP1-GST was expressed in soluble forms,and the VP1-His was found to be expressed in inclusion body.The VP1-His protein was renatured by dialysis renatration.Both fusion proteins were reached purities of more than 90%.High titer antiserum was obtained by immunizing New Zealand white rabbits.Conclusion Both the expressed fusion proteins showed high immunogenicity and antigenicity,and the prepared polyclonal antibody showed high specificity,which laid a foundation of development of detection method for Echovirus.更多还原