甲型H1N1流感病毒NS1蛋白在大肠杆菌中的表达、抗体制备及在血清学检测中的应用

发布时间：2018-03-19 18:49

本文选题：流感病毒　切入点：非结构蛋白(NS1)　出处：《南京医科大学》2009年硕士论文　论文类型：学位论文

【摘要】：流感病毒属正粘病毒科,有包膜、分节段的负单链RNA病毒。流感病毒分为甲、乙、丙三型。甲型流感病毒可分为16个HA亚型和9个NA亚型。上个世纪爆发四次甲型流感大流行,即1918年西班牙流感(H1N1)、1957年亚洲流感(H2N2)、1968年香港流感(H3N2)和1977年重现的H1N1流感。1997年香港爆发高致病性禽流感(H5N1)感染人。截至2009年5月6日,WHO共报道了423例感染,258例死亡,死亡率高达61.0%。2009年4月墨西哥爆发甲型H1N1流感,经过短暂的时间,全球33个国家正式报告了5728例流感感染病例。这种病毒已被证实经由人-人形式传播。WHO预警级别已升至5级。流感病毒容易发生抗原突变和基因重组,即抗原漂移和抗原转换,产生新的病毒株甚至新的亚型。猪、狗等家畜亦可作为流感的贮存宿主,禽流感和人流感病毒基因也可在其体内发生重组。并且由于野生鸟类与家禽的接触和迁徙,这都加快了流感病毒基因的重组与变异。因此对流感病毒的全球检测非常重要。流感也是第一个全球监控的疾病。但由于流感疫苗的广泛使用,进行流感监测时必须区分出疫苗免疫动物和病毒感染动物(DIVA)。为此WHO提出和建立了多种DIVA策略,包括哨兵家禽策略、亚单位疫苗策略、异源神经氨酸酶策略和非结构蛋白(NS1)策略等。NS1检测的最大优势是适用于任何一种甲型流感病毒且只需一种检测方法,而不受病毒抗原漂移的影响。NS1法较其它几种方法更快速、方便和经济。流感的最有效控制手段是疫苗接种,对人高致病性禽流感疫苗研究的一个方向是发现流感通用型疫苗,即寻找病毒保守且与病毒致病性密切相关的蛋白。这类抗原不能阻止病毒感染宿主,但可减轻所有的甲型流感病毒的病情,减低死亡率。这类疫苗对高致病性流感病毒的预防显得更有意义。在所有甲型流感病毒中,NS1蛋白高度保守,并与流感病毒致病性密切相关。目前关于NS1蛋白免疫保护作用研究的报道不多且结论也不相一致。本试验主要研究内容和结果如下: 一、流感病毒NS1蛋白在大肠杆菌中的表达、纯化及多克隆抗体制备设计NS1特异性引物,克隆NS1全长序列,构建重组原核表达质粒pET28a-NS1。重组质粒经PCR、酶切鉴定、核酸序列测定和分析后转化大肠杆菌BL21,经诱导表达的融合蛋白用镍柱亲和层析纯化,SDS-PAGE和Western blot结果显示表达的蛋白分子量约为26 kDa。NS1免疫新西兰兔后可获得高效价多克隆抗体(105以上),并可用于Western blot检测。二、建立NS1-ELISA检测方法以纯化NS1蛋白包被96孔板,建立检测小鼠血清NS1抗体的间接ELISA方法,在病毒感染后15-30d可正确区分出病毒感染动物和疫苗免疫动物。此方法具有较高的敏感性(94.4%)和特异性(88.9%)。三、NS1蛋白免疫保护作用研究 NS1纯化蛋白两次免疫小鼠后,第30d小鼠抗体阳性率为84.6%。NS1免疫小鼠的生存率为37.5%,高于病毒对照组(16.0%),但远低于疫苗免疫组(81.3%)。半数生存时间、肺病变和肺指数等指标较病毒对照组没有显著差异,说明NS1蛋白不是流感的主要保护性蛋白,NS1蛋白对小鼠免疫保护作用较差。综上所述,本研究成功克隆了流感病毒NS1基因并在大肠杆菌中表达,并将此蛋白初步运用于病毒感染小鼠和疫苗免疫小鼠的血清学鉴别检测。NS1蛋白对小鼠的免疫保护作用较差。本实验制备的高效价NS1多克隆抗体可用于Western blot检测,为今后研究NS1的生物学活性奠定了基础。
[Abstract]:Influenza virus family Orthomyxoviridae envelope, segmented negative strand RNA virus. Influenza viruses are divided into a, B, C three. Influenza A virus can be divided into 16 subtypes of HA and 9 NA subtypes. The last century the outbreak of the four influenza pandemic, the 1918 Spanish flu (i.e. H1N1), the 1957 Asian flu (H2N2), influenza A (H3N2) in Hongkong in 1968 and 1977 to reproduce the H1N1 influenza.1997 in Hongkong outbreak of highly pathogenic avian influenza (H5N1) infection. As of May 6, 2009, WHO reported 423 cases of infection, 258 cases died, the mortality rate as high as 61.0%.2009 in April Mexico outbreak of influenza a H1N1. After a short time, the 33 countries in the world have officially reported 5728 cases of influenza infection. The virus has been confirmed by the people who form the spread.WHO warning level has risen to 5.
The influenza virus antigen prone to mutation and gene recombination, namely antigenic drift or antigenic shift, produce new strains and new subtype. Pigs, dogs and other animals can be used as a reservoir of influenza, avian flu and human influenza virus gene recombination can also occur in its body. And by the Yu Yesheng birds and poultry contact and this migration, are speeding up the reorganization and variation of influenza virus gene. So the detection of global influenza virus is very important. The flu is also the first global monitoring of disease. But due to the widespread use of influenza vaccine, influenza surveillance must distinguish vaccinated animal and animal virus infection (DIVA). Therefore WHO is proposed in this paper a variety of DIVA strategy, including the sentinel poultry subunit vaccine strategy, strategy, strategy and non structural protein heterologous neuraminidase (NS1) the biggest advantage strategy of.NS1 detection is applicable to any Influenza A virus and only one detection method, without the effect of virus antigen drift,.NS1 method is more rapid, convenient and economical than the other methods.
The most effective means of control of influenza vaccine, a direction of highly pathogenic avian influenza vaccine research is to find a universal flu vaccine for the virus, namely conservative and virus pathogenicity related proteins. These antigens can prevent virus infection, but can reduce all influenza A virus disease reduce mortality., to prevent this kind of vaccine against highly pathogenic influenza virus is more important. In all influenza A virus, NS1 protein is highly conserved, and the pathogenicity of influenza virus NS1 protein are closely related. The immune protective effects of the report is not much and the conclusions are not consistent.
The main contents and results of this experiment are as follows:
A, expression of influenza virus NS1 protein in Escherichia coli, purification and preparation of polyclonal antibody for NS1 specific primer, the full-length NS1 clone, recombinant expression plasmid of recombinant pET28a-NS1. plasmid by PCR, enzyme digestion, DNA sequencing and analysis after transformed into Escherichia coli BL21, the inducible expression of the fusion protein by nickel column affinity chromatography, SDS-PAGE and Western blot showed that the molecular weight of the expressed protein was about 26 kDa.NS1 New Zealand rabbits were immunized after high titer polyclonal antibody (more than 105), and can be used for Western blot detection.
Two, the establishment of NS1-ELISA detection method
The purified NS1 protein was coated with 96 porous plates, and an indirect ELISA method was established to detect NS1 antibody in mice serum. After virus infection, 15-30d can correctly distinguish virus infected animals and vaccine immunized animals. This method has high sensitivity (94.4%) and specificity (88.9%).
Three, study on the protective effect of NS1 protein
The NS1 protein was purified two times after immunization, the positive rate of 30d antibody in mice 84.6%.NS1 mice survival rate was 37.5%, higher than that of the virus control group (16%), but much lower than vaccine group (81.3%). The median survival time, there is no significant difference between lung disease and lung index compared with the virus control group, the main protective protein NS1 protein is not the flu, poor NS1 protein on mice immune. In summary, this study successfully cloned the NS1 gene of influenza virus and its expression in Escherichia coli, and
The initial use of this protein to detect.NS1 protein in serum is poor in mice infected with the virus and vaccine immune protection to mice. The experimental preparation of high titer NS1 polyclonal antibody can be used for Western blot detection, which laid the foundation for the future research on biological activity of NS1.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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