戊型肝炎病毒ORF2蛋白与新型融合标签Halo Tag的融合表达 |
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引用本文: | 马天武,黄芬,井申荣,曾韦锟,禹文海.戊型肝炎病毒ORF2蛋白与新型融合标签Halo Tag的融合表达[J].粉末涂料与涂装,2012,25(12):1583-1586. |
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作者姓名: | 马天武 黄芬 井申荣 曾韦锟 禹文海 |
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作者单位: | 1. 昆明理工大学生命科学与技术学院 昆明 650500 2. 中国医学科学院北京协和医学院 医学生物学研究所 医学灵长类中心实验动物部 昆明 650118 |
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基金项目: | 云南省教育厅基础研究面上项目(项目编号:2011Y382) |
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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。
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关 键 词: | 肝炎病毒 戊型 ORF2蛋白 HaloTag 融合蛋白 原核细胞 基因表达 |
Fusion expression of hepatitis E virus ORF2 protein with Halo Tag fusion tag |
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Abstract: | 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. |
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Keywords: | Hepatitis virus E ORF2 protein Halo Tag Fusion protein Prokaryotic cells Gene expression |
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