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目的在大肠杆菌KRX中表达戊型肝炎病毒(Hepatitis E virus,HEV)ORF2与新型融合标签Halo Tag融合蛋白。方法采用RT-PCR法从4型HEV毒株中扩增ORF2基因部分片段(aa 382~620),插入含新型融合标签Halo Tag的原核表达载体pFN18the中,构建重组表达质粒pFN18the-ORF2,转化大肠杆菌KRX,鼠李糖诱导表达Halo-ORF2融合蛋白,纯化后Western blot分析融合蛋白的反应原性。结果经RT-PCR扩增出717 bp的目的基因片段;重组表达质粒pFN18the-ORF2经双酶切和测序鉴定构建正确;表达的融合蛋白Halo-ORF2相对分子质量为57 900,表达量约占菌体总蛋白的15%,以包涵体形式表达,纯化后纯度达90%以上,可与HEV IgG阳性血清特异性结合。结论在大肠杆菌KRX中表达了Halo-ORF2融合蛋白,为HEV结构蛋白抗原表位的筛选及戊肝血清学诊断的研究奠定了基础。
Objective To express fusion protein Halo Tag fusion protein of Hepatitis E virus (HEV) ORF2 in E.coli KRX. Methods A partial ORF2 gene fragment (aa 382-620) was amplified by RT-PCR from type 4 HEV strains and inserted into the prokaryotic expression vector pFN18the containing the novel fusion tag Halo Tag. The recombinant plasmid pFN18the-ORF2 was transformed into the large intestine Bacillus KRX, rhamnose induced the expression of Halo-ORF2 fusion protein, Western blot analysis of the purified fusion protein after purification. Results The 717 bp target gene fragment was amplified by RT-PCR. The recombinant plasmid pFN18the-ORF2 was identified by double enzyme digestion and sequencing. The relative molecular mass of the fusion protein Halo-ORF2 was 57 900, Fifteen percent of the total body protein is expressed in inclusion bodies. The purified protein has a purity of more than 90% and can specifically bind to HEV IgG positive sera. Conclusion Halo-ORF2 fusion protein was expressed in Escherichia coli KRX, which laid the foundation for the screening of epitopes of HEV structural proteins and serological diagnosis of hepatitis E virus.