头茎互换荧光报告流感病毒在广谱流感中和抗体筛选和检测中的应用

发布时间：2019-01-08 14:34

【摘要】：季节性流感和大流行给人类的健康带来巨大威胁。流感病毒是单股负链RNA病毒,属于正粘科,分为三种类型,其中A型流感最为广泛,危害最大。流感病毒表面血凝素(HA)分子是主要抗原,三维结构为三聚体结构,52-277位保守的半胱氨酸将血凝素分子分为一个球状头部和杆状茎部两部分。HA球头部含有唾液酸受体结合位点,介导病毒与受体细胞的结合,是诱导机体产生中和抗体的主要靶标。不同亚型HA头部结构域氨基酸不同,高度可变,针对HA球头部的抗体特异性较强。HA杆状茎部在所有HA亚型中相对保守,可诱导产生广谱中和抗体,但由于茎部抗原表位的暴露较差,该类抗体的制备和筛选较为困难。目前广谱中和抗体的研究与制备是流感抗体研究中的热点。然而,传统的血凝抑制与微量中和实验很难快速筛选检测出针对HA茎部保守区域的抗体。利用反向遗传技术,制备含有不同HA头部和茎部的重组荧光报告流感病毒,通过头茎互换HA流感荧光报告病毒的次序筛选,将能快速筛选得到针对保守茎部的广谱中和抗体。前期我们实验室成功拯救出一株携带Gaussia.luciferase荧光报告基因的流感病毒,命名为IAV-luc。IAV-luc是复制完全型病毒,能够在感染的细胞和动物体内稳定表达荧光素酶。通过荧光素酶的检测,能够直观、快速的检测报告病毒的复制情况。本研究尝试在IAV-luc背景下拯救携带不同亚型头茎互换的HA荧光报告病毒,利用HA头茎互换荧光流感病毒靶向HA茎部结构域,开发一种高效、便捷、灵敏的高通量广谱中和抗体筛选方法。本研究中,成功拯救出一系列的荧光流感病毒 H1-PR8/H1-CA09-luci,H1-PR8/H5-HK-luci,H9-HK/H1-PR8-luci,H1-CA09/H1-PR8-Luci,将头茎互换荧光流感病毒与待测抗体孵育后,将混合液转移至细胞培养板,培养24h后加入腔肠素底物检测报告病毒的荧光素酶的活性。荧光素酶活性的强弱直接反映出待检测抗体对报告病毒的抑制程度,我们可以判断出待检抗体的结合部位及中和活性。本研究初步使用已知中和抗体4E6、CR9114、C179、8852等验证头茎互换荧光流感病毒的识别能力。结果显示,4E6只能中和 H5-HK-luci,不能中和 H1-PR8/H5-HK-luci,而 CR9114、C179、8852对 H1-PR8-luci、H5-HK97-luci、H1-PR8/H5-HK 有中和活性,证实了 4E6 抗体识别H5-HA的头部,CR9114、C179和8852则是识别茎部的广谱中和抗体。综上,本研究建立一种通过HA头茎互换荧光报告流感病毒快速检测茎部中和抗体的实验方法。该方法首次将荧光素酶基团引入头茎互换流感病毒中,大大提高针对HA茎部的广谱中和抗体检测的效率和灵敏度,为高通量筛选广谱流感中和抗体提供可行的方案,为流感病毒的防治与流行病学调查提供新的思路。
[Abstract]:Seasonal influenza and pandemics pose a great threat to human health. Influenza virus is a single negative strand RNA virus, which belongs to orthomycology and is divided into three types. Type A influenza virus is the most widespread and harmful. The hemagglutinin (HA) molecule on the surface of influenza virus is the main antigen, and the three-dimensional structure is trimer structure. The conserved cysteine at the 52-277 position divides the hemagglutinin molecule into a spherical head and a rod-shaped stem. The head of the HA ball contains sialic acid receptor binding sites that mediate the binding of the virus to the receptor cells. It is the main target that induces the body to produce neutralizing antibody. The amino acids of different subtypes of HA head domain are different and highly variable, and the specificity of antibodies against the head of HA spheres is strong. The stem of HA rod is relatively conserved in all HA subtypes, which can induce the production of broad-spectrum neutralizing antibodies. However, because of the poor exposure of stem epitopes, it is difficult to prepare and screen these antibodies. At present, the research and preparation of broad spectrum neutralizing antibody is a hot spot in influenza antibody research. However, traditional hemagglutination inhibition and microneutralization experiments are difficult to quickly screen and detect antibodies against the conserved region of the stem of HA. Reverse genetic technique was used to prepare recombination fluorescent report influenza virus containing different HA heads and stems. By selecting the HA fluorescent report virus from head to stem, broad spectrum neutralizing antibodies against conserved stems could be obtained quickly. Earlier, our laboratory successfully rescued a influenza virus carrying the Gaussia.luciferase fluorescent reporter gene, named IAV-luc.IAV-luc, which is a replica complete virus that can stably express luciferase in infected cells and animals. The detection of luciferase can detect the replication of virus directly and quickly. In this study, we try to rescue the HA fluorescent reporter virus carrying different subtypes of capillaries in the background of IAV-luc, and use the HA capillaries to target the HA stem domain to develop an efficient and convenient one. A sensitive method for screening high throughput broad-spectrum neutralizing antibodies. In this study, we succeeded in saving a series of fluorescent influenza viruses H1-PR8 / H1-CA09-lucio H1-PR8 / H5-HK-lucignan H9-HKR / H1-PR8-luci-H1-CA09 / H1-PR8-Luci, After incubating the head stem exchange fluorescent influenza virus with the antibody to be tested, the mixture was transferred to the cell culture plate. After 24 hours of culture, the luciferase activity of the reported virus was detected by the addition of an enterin substrate. The activity of luciferase can directly reflect the inhibition degree of antibody to report virus, and we can judge the binding site and neutralization activity of antibody to be detected. In this study, we preliminarily used the known neutralizing antibody 4E6C9114C179T8852 to verify the recognition ability of the capillary-stem exchange fluorescent influenza virus. The results showed that 4E6 could only neutralize H5-HK-luci-luciand could not neutralize H1-PR8 / H5-HK-luci.And CR9114,C179,8852 had neutralizing activity for H1-PR8-luci-H5-HK97-luci-H1-PR8 / H5-HK, which confirmed that 4E6 antibody recognized the head of H5-HA and CR9114,. C179 and 8852 are broad-spectrum neutralizing antibodies that recognize stems. In conclusion, an experimental method for rapid detection of neutralizing antibodies in stems by HA head-stem exchange fluorescence was established. The luciferase group was introduced into the head stem swap influenza virus for the first time, which greatly improved the efficiency and sensitivity of the detection of broad-spectrum neutralizing antibody against HA stem, and provided a feasible scheme for high-throughput screening of broad-spectrum influenza neutralizing antibody. To provide a new idea for the prevention and control of influenza virus and epidemiological investigation.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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