H5N1亚型流感病毒血凝素(HA)蛋白的表达鉴定及免疫原性研究  

Expression,identification,and immunological study of H5N1 influenza virus hemagglutinin(HA)protein

作　　者：孟令楠[1,2] 任志广[3] 冀显亮[2,4] 于竟杰 李元果[2,5] 李胜楠[1,2] 于志君[2,6] 孙伟阳[2] 国娇[1,2] 赵永坤[2] 王铁成[2] 冯娜[2] 王化磊[2] 杨松涛[2] 高玉伟[2] 夏咸柱[2] 胡桂学[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130062 [3]河南大学医学院,河南开封475004 [4]内蒙古农业大学,内蒙古呼和浩特010010 [5]吉林大学,吉林长春130000 [6]北京协和医学院,北京100730

出　　处：《中国病原生物学杂志》2016年第3期209-214,共6页Journal of Pathogen Biology

基　　金：公益性行业(农业)科研专项基金项目(No.201303042)

摘　　要：目的为了获得H5N1HA蛋白,利用昆虫杆状病毒表达系统表达了A/meerkat/Shanghai/SH-1/2012(clade2.3.2.1)的HA基因并获得重组H5N1亚型流感病毒HA蛋白。方法将H5N1亚型2.3.2.1谱系的HA蛋白基因扩增并克隆到pFastBacⅠ载体中,构建重组穿梭质粒,转染昆虫sf9细胞后获得重组杆状病毒,对大量培养后表达的H5N1HA蛋白进行SDS-PAGE、Western blot和间接免疫荧光检测。分别在0d和14d用纯化的表达产物HA蛋白免疫小鼠,免疫两周后进行H5N1攻毒试验,记录小鼠体重变化率及生存率。结果测序鉴定重组表达载体构建正确;SDS-PAGE显示纯化的表达蛋白分子质量为65ku;Western blot分析该蛋白能够被兔抗流感病毒IgG识别;鸡抗流感病毒抗体间接免疫荧光法测定HA蛋白的表达,在感染HA的昆虫细胞中出现特异性荧光;攻毒试验表明HA蛋白能够在小鼠体内诱导产生免疫应答并且保护H5N1流感病毒的致死性攻击。结论成功的表达了H5N1的HA蛋白,并且此HA蛋白能成功的诱导小鼠体内的免疫反应,并起到良好的保护作用。Objective A baculovirus expression system was used to express the hemagglutinin（HA）protein of A/meerkat/Shanghai/SH-1/2012（clade 2.3.2.1）influenza virus in order to acquire the HA protein of H5N1. MethodsThe HA gene of the H5N1 avian influenza virus was amplified and cloned into pFastBacI.A shuttle plasmid containing the target gene was constructed using homologous recombination technology.A recombinant baculovirus containing the HA gene was rescued by transfecting sf9 insect cells with shuttle plasmids.The HA protein was identified with SDSPAGE,Western blotting,and indirect immunofluorescence.To evaluate the immunogenicity of HA,mice were immunized twice with the purified HA protein on day 0and 14.Two weeks after the 2rounds of immunization,mice were challenged with the H5N1 virus.All mice were observed daily to monitor changes in body weight and mortality. Results In reducing SDS-PAGE,the purified protein produced a protein band that was consistent with a molecular mass of 65ku;a Western blot assay indicated that the purified HA protein was specifically recognized by rabbit IgG antiserum.Chicken antiserum against the H5N1 virus was used to determine the level of HA expression using IFA.Specific fluorescence wasobserved in insect cells infected with the recombinant baculovirus containing HA.The HA protein protected mice against lethal challenges with A/meerkat/Shanghai/SH-1/2012（clade 2.3.2.1）.Results suggested that HA of H5N1 was correctly expressed and purified. Conclusion The HA protein of the H5N1 avian influenza virus was correctly expressed and it successfully induced an effective immune response in mice.

关 键 词：流感病毒 H5N1HA 疫苗 杆状病毒表达系统 

分 类 号：R373.4[医药卫生—病原生物学]