| H5N1禽流感病毒血凝素和神经氨酸酶基因在杆状病毒中的共表达 |
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| 引用本文： | 刘丽琦,余慧燕,于在江,等. H5N1禽流感病毒血凝素和神经氨酸酶基因在杆状病毒中的共表达[J]. 实用预防医学, 2014, 0(5): 543-546 |
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| 作者姓名： | 刘丽琦 余慧燕 于在江 等 |
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| 作者单位： | [1]中国疾病预防控制中心病毒病预防控制所; [2]世界卫生组织流感参比和研究合作中心,北京102206 |
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| 摘 要： | 目的构建共表达H5N1禽流感病毒血凝素(HA)和神经氨酸酶(NA)蛋白的杆状病毒表达系统。方法利用PCR方法分别扩增A/Hubei/1/2010(H5N1)病毒的HA和NA基因,克隆至经改造带有对虾白斑综合病毒(WSSV)早期启动子iel的mpFastBac Dual载体,构建mpFast Bac-HA-NA表达质粒,转化DH10Bac感受态细胞,用获得的Bacmid-HA-NA穿梭质粒转染sf9细胞,获得重组杆状病毒Bac-HA-NA;提取重组杆状病毒Bac-HA-NA的DNA并使用PCR方法检测;利用Western blot检测HA和NA表达,并分别检测重组杆状病毒Bac-HA-NA红细胞凝集能力和神经氨酸酶活性。结果经PCR鉴定,构建的质粒Bacmid-HA-NA正确;Western blot检测表明Bacmid-HANA能在sf9细胞中有效表达HA和NA蛋白,生物活性检测表明Bac-HA-NA能引起红细胞凝集并具有神经氨酸酶活性。结论获得了能同时表达禽流感病毒HA和NA的重组杆状病毒Bac-HA-NA,为进一步研究流感疫苗奠定了基础。
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| 关 键 词： | 禽流感病毒 血凝素 神经氨酸酶 杆状病毒表达系统 共表达 |
Co-expression of hemagglutinin and neuraminidase genes from the avian influenza virus H5N1 in baculovirus-expression system |
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| Affiliation: | LIU Li - qi, YU Hui -yan, YU Zai - jiang, SHU Yue - long (National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China) |
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| Abstract: | Objective To construct the baculovirus - expression system co- expressing hemagglutinin （HA） and neuramini- dase （NA） genes of avian influenza virus H5N1. Methods HA and NA genes of A/Hubei/1/2010 （H5N1） virus were ampli- fied by PCR, and then inserted into modified mpFastBac Dual vector, which contained early promoter of white spot syndrome vir- us （WSSV）, to construct mpFastBac - HA- NA expression plasmid. Recombinant shuttle vector Bacmid - HA- NA was ob- tained after transforming DH10Bac competent cells. After transfecting into sf9 cells, recombinant baculovirus Bac- HA- NA was obtained. The Bac - HA- NA genome was extracted and identified by PCR. HA and NA proteins expressed by recombinant bac- ulovirus - system were determined by Western bolt. Biological activities of Bac - HA- NA were determined by hemagglutination test and neuraminidase activity detection. Results PCR proved that recombinant shuttle vector Bacmid - HA- NA was suc- cessfully constructed. Western blot test showed that HA and NA proteins could be expressed simultaneously on the surface of recombinant baculovirus and biological activity tests suggested that Bac - HA- NA sustained their hemagglutination activities and neuraminidase activities. Conclusions Recombinant baculovirus Bac- HA- NA simultaneously expressing HA and NA genes of H5N1 influenza virus is successfully obtained. This paper provides a basis for further study on novel development of influenza |
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| Keywords: | Avian influenza Hemagglutinin Neuraminidase Baculovirus expression system Co-expression |
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