摘要
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 hepatitis E virus（HEV）ORF2 protein with Halo Tag fusion tag in E.coli KRX strain.Methods Partial ORF2 gene（aa 382 ~ 620）was amplified from HEV type 4 by RT-PCR and inserted into prokaryotic expression vector pFN18the containing Halo Tag fusion tag.The constructed recombinant plasmid pFN18the-ORF2 was transformed to E.coli KRX and induced with rhamnose.The expressed Halo-ORF2 fusion protein was purified and analyzed for reatogenicity by Western blot.Results The target gene fragment at a length of 717 bp was amplified by RT-PCR.Restriction analysis and sequencing proved that recombinant plasmid pFN18the-ORF2 was constructed correctly.The expressed fusion protein,with a relative molecular mass of 57 900,contained about 15% of total somatic protein and reached a purity of more the 90% after purification,which showed specific binding to HEV IgG positive serum.Conclusion Recombinant Halo-ORF2 fusion protein was expressed in E.coli KRX,which laid a foundation of screening of antigenic epitope of structural protein of HEV and study on serological diagnosis of HE.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第12期1583-1586,共4页
Chinese Journal of Biologicals
基金
云南省教育厅基础研究面上项目(2011Y382)
