4种血清型登革病毒多表位重组抗原的表达及血清学初步评价

发布时间：2018-02-09 09:02

本文关键词： 登革病毒抗原蛋白原核表达血清学评价　出处：《中国人兽共患病学报》2017年01期 　论文类型：期刊论文

【摘要】：目的在原核表达系统中对登革病毒包膜蛋白(E蛋白)和非结构蛋白1(NS1)融合的B细胞抗原表位进行表达、纯化及血清学评价。方法将B细胞抗原表位用形成α螺旋的连接肽(EAAAK)2作为接头,串联合成1条全新的多表位融合重组基因rE,将其克隆到原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3)后经IPTG诱导表达融合蛋白,用镍柱对重组蛋白纯化,并用SDS-PAGE和Western blot方法鉴定表达产物。以融合蛋白为抗原,用间接ELISA检测登革热病人血清IgM抗体。结果重组表达载体pET28a-rE构建成功,并在大肠杆菌BL21(DE3)中成功表达。融合蛋白主要以包涵体形式存在,用镍柱纯化获得高纯度的目的蛋白,SDS-PAGE和Western blot检测结果显示蛋白分子量大小与预期结果相符,建立的间接ELISA具有较高的准确性。结论原核表达的登革病毒多表位融合蛋白具有良好的血清学检测价值。
[Abstract]:Objective to express B cell antigen epitopes fused with dengue virus envelope protein (E) and nonstructural protein 1 (NS1) in prokaryotic expression system. Methods A novel multiepitope fusion recombinant gene, rEE, was synthesized from B cell antigen epitope using 伪 helix ligated peptide, AAAKN 2, as a junction. The recombinant gene was cloned into prokaryotic expression vector pET-28a(), and the recombinant gene was cloned into the prokaryotic expression vector pET-28a (pET-28a ()). Expression of the fusion protein was induced by IPTG. The recombinant protein was purified by nickel column and identified by SDS-PAGE and Western blot. The fusion protein was used as antigen. Results the recombinant expression vector pET28a-rE was successfully constructed and successfully expressed in E. coli BL21DE3. The fusion protein was mainly in the form of inclusion body. The results of SDS-PAGE and Western blot analysis showed that the molecular weight of the protein was in accordance with the expected results. Conclusion the polyepitope fusion protein expressed in prokaryotic cells has good serological value.
【作者单位】：广东医科大学检验医学研究所;东莞市第八人民医院;
【分类号】：R373.33

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