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AIVNA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.coliDH10B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1 352 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。AIVNA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AIVNA基因在昆虫Sf9细胞中表达成功。
[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 1 352 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.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第7期28-32,共5页
Journal of Northwest A&F University(Natural Science Edition)
