| 摘要: |
| 【目的】 在Sf9昆虫细胞中表达H5N1亚型禽流感病毒(Avian influenza virus,AIV)神经氨酸酶（NA）基因。【方法】 用PCR方法克隆H5N1亚型AIV NA基因，定点插入到转移载体pFastBacHTb中，构建重组转移载体pFastBacHTb-NA。将其转化到含有AcBacmid和helper质粒的E.coli DH10B感受态细胞中，与AcBacmid重组，获得rAcBacmid-NA。提取重组的Bacmid，转染处于对数生长期的昆虫Sf9细胞，获得重组杆状病毒vAc-NA。将vAc NA重新感染处于对数生长期的昆虫Sf9细胞，3~5 d后收集被感染细胞。取转染细胞、感染细胞、破碎感染细胞上清和破碎感染细胞沉淀进行SDS-PAGE 和Western-blotting 分析。【结果】 通过PCR成功地克隆了1352 bp 的H5N1亚型AIV不含信号肽序列的NA基因；pFastBacHTb-NA和rAcBacmid-NA构建成功。AIV NA基因在昆虫Sf9细胞内得到有效表达，表达产物分子质量约为52 ku，且具有免疫原性。【结论】 H5N1亚型AIV NA基因在昆虫Sf9细胞中表达成功。 |
| 关键词: H5N1亚型禽流感病毒 神经氨酸酶 杆状病毒载体 真核表达 |
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| Expression of influenza virus subtype H5N1 neuraminidase gene in Sf9 insect cells |
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| Abstract: |
| 【Objective】 The research studied influenza virus subtype H5N1 neuraminidase (NA) gene expressed in Sf9 insect cells.【Method】 NA,the neuraminidase (NA) gene of H5N1 subtype avian influenza virus (AIV),was successfully amplified by polymerase chain reaction (PCR).The gene was inserted into plasmid pFastBacHTb to obtain recombinant transfer vector pFastBacHTb-H5N1-NA. pFastBacHTb-H5N1-NA was transformed into DH10B cells carrying both AcBacmid and helper plasmid.Recombinant bacmid rAcBacmid-H5N1-NA with the insertion of NA gene was obtained by specific transposition between pFastBacHTb-H5N1-NA and AcMNPV bacmid.Recombinant virus vAc-H5N1-NA was obtained by transfecting rAcBacmid-H5N1-NA into Sf9 insect cells.Supernatant containing vAc-H5N1-NA was collected and Sf9 cells were infected by vAc-H5N1-NA.Cells collected 3-5 days after transfection and infection were broken by the sonicator.Then the supernatant and the sediment were detected by SDS-PAGE and Western-blotting.【Result】 NA,the neuraminidase (NA) gene of H5N1 subtype avian influenza virus (AIV),was successfully amplified by PCR,which was 1352 bp.Recombinant transfer vector pFastBacHTb-H5N1-NA was reconstructed and recombinant rAcBacmid-NA was obtained.NA protein NA was highly expressed in Sf9 insect cells with the approximate molecular weight 52 ku,and with immunogenicity.【Conclusion】 Influenza virus subtype H5N1 neuraminidase gene (NA) was expressed in Sf9 insect cells successfully. |
| Key words: H5N1 subtype avian influenza virus neuraminidase baculovirus vector eukaryotic expression |
