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H5N1亚型高致病性禽流感病毒NP基因在昆虫细胞
中的表达及其表达产物的生物学活性
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万春和，刘明*，刘春国，杨涛，刘大飞，齐金龙，陈浩，杜金铃，李洪涛
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（中国农业科学院 哈尔滨兽医研究所 农业部动物流感重点开放实验室
兽医生物技术国家重点实验室，黑龙江 哈尔滨 150001）
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摘要：
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根据GenBank上登录的H5N1亚型高致病性禽流感病毒(HPAIV)核蛋白（NP）基因序列设计引物，通过PCR扩增出A/Goose/HLJ/QFY/04(H5N1)毒株的NP基因片段，将其克隆到pFastBacGP67B杆状病毒载体上，将筛选的阳性重组转座载体pFastBacGP67B-NP转化至含有杆状病毒穿梭载体的DH10Bac感受态细胞，经蓝白斑筛选和PCR鉴定，构建了杆状病毒表达载体，获得了重组转座子（rBacmid-NP），在脂质体介导下转染对数生长期的Sf9昆虫细胞，获得重组杆状病毒(rBv-NP)。SDS-PAGE分析、Western-blotting检测、免疫组化分析和ELISA试验结果表明，分子质量约为54ku的重组核蛋白得到了高效表达，该表达蛋白具有良好的生物学活性。 |
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关键词：
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禽流感病毒；核蛋白基因；昆虫细胞杆状病毒表达 |
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中图分类号：S 852.659.5
文献标识码：
A 文章编号：1673-4696(2008)05-0380-06 |
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Expression of NP gene of highly pathogenic avian influenza virus subtype
H5N1 in insect cells and identification of biological activities of
the expressed product |
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WAN Chun-he,LIU Ming,LIU Chun-guo,YANG Tao,LIU Da-fei,QI Jin-long,CHEN Hao,
DU Jin-ling,LI Hong-tao |
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（National Key Laboratory of Veterinary Biotechnology/ Key Laboratory of Animal Influenza of the Ministry of
Agriculture/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences，Harbin 150001，China） |
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Abstract: A pair of primers were designed and synthesized according to the nucleoprotein(NP) gene sequence of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 available in GenBank.The NP gene of HPAIV was amplified by PCR from A/Goose/HLJ/QFY/04(H5N1) strain and then cloned into the baculovirus vector pFastBacGP67B.The recombinant positive plasmid pFastBacGP67B-NP was identified by digestion with restriction enzyme and sequencing.The purified plasmid DNA was transformed into competent cells of Escherichia coli DH10Bac containing baculovirus shuttle vector for transposition into the bacmid and the positive colonies were screened by blue/white spot selection.Recombinant bacmid (rBacmid-NP) purified DNA were verified by PCR analysis and transfected into Sf9 insect cells during log growth phase using Cellfection reagent to obtain the production of recombinant viral stock(rBv-NP).The expressed NP protein was analyzed by SDS-PAGE,Western-blotting,immunohistochemistry and ELISA tests,and the results demonstrated that recombinant NP protein of 54ku in molecular mass was expressed in high-level and had good biological activity. |
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Key words: avian influenza virus;NP gene;baculovirus insect cell system |
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