H5N1血凝素蛋白的重组牛痘病毒表达纯化以及耐奥司他韦神经氨酸酶动力学研究

发布时间：2018-01-07 20:42

本文关键词：H5N1血凝素蛋白的重组牛痘病毒表达纯化以及耐奥司他韦神经氨酸酶动力学研究　出处：《中南大学》2009年博士论文　论文类型：学位论文

【摘要】： 高致病性禽流感病毒H5N1能够打破宿主限制“禽传人”,造成人感染禽病毒H5N1的病例。禽流感病毒H5N1尚不具备“人传人”的能力。宿主限制主要与血凝素受体结合特性有关。近期基因分析研究表明,某些人禽流感病毒H5N1在受体结合部位也有突变。本研究第一部分旨在从HA晶体结构解析,进一步推测H5N1病毒HA对宿主细胞受体结合特性的改变,可否使禽流感病毒H5N1具备“人传人”的能力。首先用定点突变技术在H5 HA受体结合部位或临近位点引入目的氨基酸变异A134V、I151F、E186D和I151F+A134V+E186D。再通过反向遗传学方法拯救带有这些变异位点的重组流感病毒,结果发现H5 A134V和H5 E186D均能在鸡胚传代中稳定表达,并且得到了这两种HA晶体,及HA-人/禽受体结合复合物的晶体X线衍射图,从而分析得出H5 E186D倾向于结合人受体而H5 A134V受体结合特性与野生型HA相同——倾向于结合禽受体。另一个变异位点H5 I151F在鸡胚传代后变成了H5 F151L,提示I151F不利于病毒在鸡胚中繁殖,可能与该位点弱化HA与鸡胚中的禽受体SAα2,3 Gal结合有关。为了进一步证实该假设,我们通过构建重组牛痘病毒表达并纯化H5 I151F和H5 I151F+A134V+E186D,首次成功获得了H5 I151F晶体,为后续开展晶体结构解析和多聚糖微阵列分析HA受体结合特性等工作,进一步研究禽流感病毒人传人的可能性打下基础。为了应对下一次流感大流行的到来,很多国家将神经氨酸酶抑制剂-奥司他韦做为一种战略储备药物来储存。随着该药物的广泛使用,奥司他韦耐药现象也日趋突显。以往的研究重点局限在N1的H274Y,H253Y和N294S。本研究中我们主要研究N1的另外两个氨基酸变异位点I117V和I134V。这两个突变首先在鸡流感病毒H5N1中被发现有降低奥司他韦药敏的作用。如果人感染了带有这些氨基酸突变的H5N1病毒,临床上还能不能用奥司他韦抗病毒治疗?为此,我们将这两个突变位点引入人感染的H5N1病毒,通过该病毒对神经氨酸酶的酶促反应动力学研究得出将这两个变异引入能感染人的H5N1 NA,并不会导致病毒对奥司他韦耐药,但仍能使病毒对奥司他韦敏感性降低,这可能与该位点临近NA活性中心的保守氨基酸E119,从而间接影响药物与NA的结合有关。
[Abstract]:Highly pathogenic avian influenza virus H5N1 can break host restrictions on "avian to human transmission". Avian influenza virus H5N1 does not have the ability of "human transmission". Host restriction is mainly related to the binding characteristics of hemagglutinin receptor. Some human avian influenza virus H5N1 also has mutations in the receptor binding site. The first part of this study was designed to analyze the crystal structure of HA. Further speculate the changes of receptor binding characteristics of H5N1 virus HA to host cells. Whether the avian influenza virus H5N1 has the ability of "human transmission". Firstly, the aim amino acid mutation A134VFI151F was introduced into the H5 HA receptor binding site or adjacent site by site-directed mutation technique. E186D and I151F A134V E186D.Rescue recombinant influenza viruses with these mutated sites by reverse genetics. The results showed that both H5A134V and H5E186D could be expressed stably in the passage of chicken embryos, and the X-ray diffraction patterns of these two HA crystals and the ha-human / avian receptor binding complex were obtained. Thus, it was found that H5 E186D tended to bind to human receptor and H5 A134V receptor had the same binding characteristics as wild type HA, which tended to bind to avian receptor. Another variant site H5 was H5. I151F was transformed into H5F151L after passage of chicken embryo. It is suggested that I151F is not conducive to the reproduction of the virus in chicken embryos, and may be related to the weakening of HA at this site with the binding of the avian receptor SA 伪 2O 3 Gal in chicken embryos. We constructed recombinant cowpox virus and purified H5I151F and H5I151F A134V E186D.The H5I151F crystal was successfully obtained for the first time. It will lay a foundation for further research on the possibility of human-to-human transmission of avian influenza virus (Avian Influenza virus) through crystal structure analysis and analysis of HA receptor binding characteristics by polyglycan microarray. In response to the next influenza pandemic, many countries store oseltamivir, a neuraminidase inhibitor, as a strategic reserve drug. The phenomenon of oseltamivir resistance is becoming more and more prominent. Previous studies have focused on N1 H274Y. H253Y and N294S. in this study, we studied two other amino acid mutation sites of N1, I117V and I134V. these two mutations were first found in avian influenza virus H5N1. Reduce the effect of oseltam. If a human is infected with the H5N1 virus with these amino acid mutations. Can still use oseltamivir antiviral treatment clinically? Therefore, we introduced these two mutation sites into the H5N1 virus infected by human, and through the kinetic study of the enzymatic reaction of the virus to neuraminidase, we found that the two mutations were introduced into the H5N1 na which could infect the human. It does not result in oseltamivir resistance, but it still reduces the sensitivity of the virus to oseltamivir, which may be associated with the conserved amino acid E119 near the na active site. This indirectly affects the combination of drugs with na.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373;R96

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