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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。
Objective To express, purify and serologically evaluate the B cell epitope fused to dengue virus envelope protein (E protein) and nonstructural protein 1 (NS1) in prokaryotic expression system. METHODS: A new multi-epitope fusion gene, rE, was cloned into the prokaryotic expression vector pET-28a (+) using the B-cell epitope peptide (EAAAK) 2 as a linker. After transformed into E. coli BL21 (DE3), the fusion protein was induced by IPTG and the recombinant protein was purified by nickel column. The expressed product was identified by SDS-PAGE and Western blot. Using the fusion protein as antigen, serum IgM antibody was detected by indirect ELISA in dengue patients. Results The recombinant expression vector pET28a-rE was successfully constructed and successfully expressed in E. coli BL21 (DE3). The fusion protein was mainly in the form of inclusion bodies. The purified protein was purified by nickel column to obtain the target protein with high purity. SDS-PAGE and Western blot showed that the molecular weight of the fusion protein was in good agreement with the expected result. Conclusion The prokaryotic expression of dengue virus multi-epitope fusion protein has good serological detection value.