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。
We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells, and purified and evaluated for its serological activities. A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide （EAAAK）2 and cloned into prokaryotic expression vector pET-28a（＋）, and transformed into E. coli BL21（DE3） cells for expression under induction of IPTG. The expressed recombinant protein was purified with 6 × His purification media, and identified by SDS-PAGE and Western blot, and its antigenicity was analyzed by using an indirect ELISA assay. The recombinant expression vector pET28a-rE was constructed and expressed in BL21 （DE3） successfully, but the re- combinant proteins mainly appeared as inclusion bodies. The target protein was obtained with high purity through the purifica- tion of affinity chromatography. SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line. The established indirect ELISA has high accuracy. This recombinant peptide antigen expressed in E. coli has good potential for serum testing..
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2017年第1期32-37,共6页
Chinese Journal of Zoonoses
关键词
登革病毒
抗原蛋白
原核表达
血清学评价
dengue virus
antigen protein
prokaryotic expression
serological evaluation
