| H3亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接ELISA检测方法的建立 |
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| 引用本文： | 万春和,刘明,刘春国,张云.H3亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接ELISA检测方法的建立[J].中国兽医科学,2010,40(3). |
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| 作者姓名： | 万春和 刘明 刘春国 张云 |
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| 作者单位： | 万春和(中国农业科学院,哈尔滨兽医研究所,兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江,哈尔滨,150001;福建省农业科学院,畜牧兽医研究所,福建,福州,350013);刘明,刘春国,张云(中国农业科学院,哈尔滨兽医研究所,兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江,哈尔滨,150001) |
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| 基金项目： | 国家"十一五"科技支撑计划项目，黑龙江省自然科学基金项目 |
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| 摘 要： | 根据GenBank上登录的H3亚型猪流感病毒HA基因序列设计了1对引物,经PCR扩增出HA基因目的片段,并将其克隆到pFastBacGP67C杆状病毒载体上,筛选阳性重组转座载体pFastBacGP67C-H3,转化至含有杆状病毒穿梭载体(bacmid)的DH10Bac感受态细胞,构建杆状病毒表达载体,获得重组转座子rBacmid-H3,在脂质体介导下转染处于对数生长期的sf9昆虫细胞,获得重组杆状病毒rBv-H3,将其感染sf9昆虫细胞,收获目的蛋白。经血凝试验、SDS-PAGE、Western-blot、免疫组织化学分析,结果表明,HA蛋白得到表达,且具有良好的免疫学活性。用表达的HA蛋白作为包被抗原,初步建立了H3亚型猪流感病毒的间接ELISA检测方法,通过对93份送检的疑似猪流感血清进行检测,结果显示,检出阳性率为46.24%。上述结果为建立快速、准确、简便的H3亚型猪流感鉴别诊断试剂盒奠定了基础。
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| 关 键 词： | H3亚型猪流感病毒 血凝素基因 昆虫细胞杆状病毒表达系统 间接酶联免疫吸附试验 |
Expression of HA gene of swine influenza virus H3 subtype in sf9 insect cells and establishment of an indirect ELISA for the detection of the virus |
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| WAN Chun-he,LIU Ming,LIU Chun-guo,ZHANG Yun.Expression of HA gene of swine influenza virus H3 subtype in sf9 insect cells and establishment of an indirect ELISA for the detection of the virus[J].Veterinary Science in China,2010,40(3). |
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| Authors: | WAN Chun-he LIU Ming LIU Chun-guo ZHANG Yun |
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| Institution: | WAN Chun-he1,2,LIU Ming1,LIU Chun-guo1,ZHANG Yun1(1.State Key Laboratory of Veterinary Biotechnology,Animal Influenza Laboratory of the Ministry of Agriculture,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China,2.Institute of Animal Science , Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China) |
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| Abstract: | Using a pair of specific primers designed according to the sequences of hemagglutinin(HA) gene of swine influenza virus(SIV) H3 subtype available in GenBank,the HA gene fragment was amplified from SIV A/Swine/Henan/S4/04(H3N2) by PCR and cloned into the baculovirus transfer plasmid pFASTBacGP67C.The recombinant pFastBacGP67C-H3 was identified by restriction enzyme and sequencing.After the transformation of DH10Bac Escherichia coli competent cells by pFastBacGP67C-H3,recombinant bacmids rBacmid-H3 was identi... |
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| Keywords: | H3 subtype SIV HA gene baeulovirus/insect cell expression system indirect ELISA |
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