本文關(guān)鍵詞：H5N1血凝素蛋白的重組牛痘病毒表達(dá)純化以及耐奧司他韋神經(jīng)氨酸酶動(dòng)力學(xué)研究　出處：《中南大學(xué)》2009年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 高致病性禽流感病毒H5N1 血凝素 反向遺傳學(xué) 重組牛痘病毒 神經(jīng)氨酸酶 奧司他韋

【摘要】： 高致病性禽流感病毒H5N1能夠打破宿主限制“禽傳人”,造成人感染禽病毒H5N1的病例。禽流感病毒H5N1尚不具備“人傳人”的能力。宿主限制主要與血凝素受體結(jié)合特性有關(guān)。近期基因分析研究表明,某些人禽流感病毒H5N1在受體結(jié)合部位也有突變。本研究第一部分旨在從HA晶體結(jié)構(gòu)解析,進(jìn)一步推測(cè)H5N1病毒HA對(duì)宿主細(xì)胞受體結(jié)合特性的改變,可否使禽流感病毒H5N1具備“人傳人”的能力。首先用定點(diǎn)突變技術(shù)在H5 HA受體結(jié)合部位或臨近位點(diǎn)引入目的氨基酸變異A134V、I151F、E186D和I151F+A134V+E186D。再通過反向遺傳學(xué)方法拯救帶有這些變異位點(diǎn)的重組流感病毒,結(jié)果發(fā)現(xiàn)H5 A134V和H5 E186D均能在雞胚傳代中穩(wěn)定表達(dá),并且得到了這兩種HA晶體,及HA-人/禽受體結(jié)合復(fù)合物的晶體X線衍射圖,從而分析得出H5 E186D傾向于結(jié)合人受體而H5 A134V受體結(jié)合特性與野生型HA相同——傾向于結(jié)合禽受體。另一個(gè)變異位點(diǎn)H5 I151F在雞胚傳代后變成了H5 F151L,提示I151F不利于病毒在雞胚中繁殖,可能與該位點(diǎn)弱化HA與雞胚中的禽受體SAα2,3 Gal結(jié)合有關(guān)。為了進(jìn)一步證實(shí)該假設(shè),我們通過構(gòu)建重組牛痘病毒表達(dá)并純化H5 I151F和H5 I151F+A134V+E186D,首次成功獲得了H5 I151F晶體,為后續(xù)開展晶體結(jié)構(gòu)解析和多聚糖微陣列分析HA受體結(jié)合特性等工作,進(jìn)一步研究禽流感病毒人傳人的可能性打下基礎(chǔ)。 為了應(yīng)對(duì)下一次流感大流行的到來,很多國家將神經(jīng)氨酸酶抑制劑-奧司他韋做為一種戰(zhàn)略儲(chǔ)備藥物來儲(chǔ)存。隨著該藥物的廣泛使用,奧司他韋耐藥現(xiàn)象也日趨突顯。以往的研究重點(diǎn)局限在N1的H274Y,H253Y和N294S。本研究中我們主要研究N1的另外兩個(gè)氨基酸變異位點(diǎn)I117V和I134V。這兩個(gè)突變首先在雞流感病毒H5N1中被發(fā)現(xiàn)有降低奧司他韋藥敏的作用。如果人感染了帶有這些氨基酸突變的H5N1病毒,臨床上還能不能用奧司他韋抗病毒治療?為此,我們將這兩個(gè)突變位點(diǎn)引入人感染的H5N1病毒,通過該病毒對(duì)神經(jīng)氨酸酶的酶促反應(yīng)動(dòng)力學(xué)研究得出將這兩個(gè)變異引入能感染人的H5N1 NA,并不會(huì)導(dǎo)致病毒對(duì)奧司他韋耐藥,但仍能使病毒對(duì)奧司他韋敏感性降低,這可能與該位點(diǎn)臨近NA活性中心的保守氨基酸E119,從而間接影響藥物與NA的結(jié)合有關(guān)。
[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 偽 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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373;R96

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本文編號(hào)：1394176