本文關(guān)鍵詞： 人腸道病毒71型 中和保護(hù)抗體 空間表位 冷凍電鏡 機(jī)制　出處：《第二軍醫(yī)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：人腸道病毒71型(enterovirus 71)屬于小RNA病毒科腸道病毒屬,是手足口病最常見的病原體。自1969年被發(fā)現(xiàn)以來,EV71已經(jīng)在全世界范圍內(nèi),尤其是亞太地區(qū)引起了數(shù)十次手足口病的大流行,造成了數(shù)千萬人感染,數(shù)萬患者產(chǎn)生嚴(yán)重的并發(fā)癥。我國是手足口病疫情的高發(fā)區(qū),2009年以來,該疾病的發(fā)病人數(shù)和死亡人數(shù)連續(xù)7年位居丙類傳染病之首。盡管現(xiàn)在EV71的疫苗完成三期臨床試驗(yàn)準(zhǔn)備上市,但是對(duì)于重癥患者,目前尚無有效的治療策略,因此研制抗病毒的特效藥物迫在眉睫。EV71病毒顆粒是一個(gè)直徑約300埃的正二十面體結(jié)構(gòu),成熟病毒衣殼由VP1、VP2、VP3和VP4組成,其中VP2、VP3和VP1的大部分區(qū)域位于病毒衣殼外部,VP4和VP1的N端位于衣殼的內(nèi)部。研究表明,在脫衣殼的過程中,病毒構(gòu)象會(huì)發(fā)生改變,5次軸處附近的“峽谷”發(fā)生坍塌形成小孔,疏水口袋內(nèi)的鞘氨醇分子釋放;2次軸處的VP2的反向平行α螺旋對(duì)會(huì)像兩側(cè)平移,形成直徑超過9埃的“孔洞”,埋藏在2次軸正下方的VP1 N端會(huì)遷移到病毒外部,參與核酸釋放通道的形成。因此篩選針對(duì)不同結(jié)合表位的單克隆抗體有助于進(jìn)一步闡述病毒脫衣殼的生物學(xué)機(jī)制。本課題采用滅活病毒和重組衣殼蛋白分別免疫Balb/C小鼠,運(yùn)用經(jīng)典的雜交瘤篩選技術(shù),獲得了15株可以特異性結(jié)合EV71病毒的單克隆抗體,其中包括6株中和保護(hù)抗體。動(dòng)物保護(hù)實(shí)驗(yàn)證明,在10ug/g乳鼠體重抗體的使用劑量下,6G5、12B6、2B6、2G12的保護(hù)效率均能達(dá)到100%,在2ug/g的抗體劑量下,6G5的保護(hù)率仍能維持100%,12B6也能達(dá)到80%的保護(hù),高于目前文獻(xiàn)所報(bào)道的中和保護(hù)性單抗。針對(duì)目前EV71可以引起無菌性腦膜炎(asepic meningitis)、腦炎(encephalitis)、腦脊髓炎(encephalomyelitis)和急性弛緩性麻痹(acute flaccid paralysis,AFP)等嚴(yán)重的神經(jīng)系統(tǒng)并發(fā)癥,我們同時(shí)也在神經(jīng)細(xì)胞上測試了15株單克隆抗體的中和保護(hù)效果,發(fā)現(xiàn)與人橫紋肌瘤細(xì)胞相比,在相同滴度病毒的攻擊下,2B6的保護(hù)效果有了大幅度的提高。究其原因,推測是因?yàn)镋V71在肌肉組織和神經(jīng)系統(tǒng)不同的入侵機(jī)制所導(dǎo)致。根據(jù)體內(nèi)外保護(hù)功能和識(shí)別表位的異同可以將中和保護(hù)抗體分為3類進(jìn)行研究,第一類2B6、2G12可以結(jié)合VP1-GHloop上的215-KQEKD-219,第二類7G6、12H3識(shí)別VP1 N端的4-VADVI-8,第三類6G5、12B6為空間表位,為鑒定6G5所識(shí)別表位,我們運(yùn)用冷凍電鏡技術(shù),解析6G5Fab與三種不同病毒生理周期的復(fù)合物結(jié)構(gòu),包括6G5Fab-天然成熟病毒,6G5Fab-空心病毒和6G5Fab-皺縮的空心病毒,分辨率分別達(dá)到了5.6埃、4.9埃和5.0埃。6G5識(shí)別位點(diǎn)位于病毒顆粒的2次軸和3次軸之間,分布在VP2和VP3的交匯處,包括VP2上135-139位的LDTKL,223-226位的GADG,296-298位的GAT和VP3上73-81位的VSAQAGKGE,144-147位的KDRA,209-212位的PNTA。這些構(gòu)成了一個(gè)由“凹槽”“把手”和“峽谷”組成的復(fù)雜的結(jié)合區(qū)域。通過突變6G5可變區(qū)上與病毒表位相互作用的關(guān)鍵氨基酸,發(fā)現(xiàn)組成該空間表位的6個(gè)功能域均參與了6G5與病毒的結(jié)合。隨后我們深入研究了三類抗體的中和保護(hù)機(jī)制,發(fā)現(xiàn)2B6的結(jié)合通過降低VP1-GHloop的柔性來穩(wěn)定5次軸附近“峽谷”底部的結(jié)構(gòu),從而抑制病毒的構(gòu)象變化,影響病毒的脫顆粒進(jìn)程;7G6可以阻礙病毒核酸通道的形成,抑制病毒核酸的釋放;最有趣的是6G5的保護(hù)機(jī)制,它可以欺騙病毒,發(fā)揮“類受體”的功能,提前介導(dǎo)細(xì)胞的脫衣殼進(jìn)程,誘導(dǎo)病毒基因組的釋放,并且抑制135S脫衣殼中間體的形成�；谝陨涎芯砍晒覀冋J(rèn)為6G5的結(jié)合位點(diǎn)是一個(gè)全新的中和表位,其保護(hù)機(jī)制為抗病毒的治療策略帶來了全新的思路,本研究成果也有助于深入理解抗EV71病毒侵染的中和保護(hù)機(jī)制,并為抗體藥物研發(fā)打下了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Human enterovirus 71 (enterovirus 71) belongs to a small RNA virus, enterovirus, is the most common pathogen of HFMD. Since 1969 it is found that EV71 has been around the world, especially the Asia Pacific region has attracted dozens of HFMD pandemic, resulting in tens of millions of people have a serious infection. The complications of tens of thousands of patients. And China is the epidemic of HFMD incidence area, since 2009, the incidence of the disease and the death toll for 7 consecutive years ranked first in class C infectious disease. Although the vaccine EV71 completed phase three clinical trials for listing, but in severe cases, there is no effective treatment strategies, therefore to develop powerful antiviral drugs of imminent.EV71 virus particles is a diameter of about 300 angstrom is twenty structure, mature capsid by VP1, VP2, VP3 and VP4, VP2, VP3 and VP1 of the region a In the viral capsid external, internal VP4 and VP1 N terminal is in the capsid. The results show that in the process of uncoating of the virus conformation changes, 5 axis near the "Canyon" collapsed to form small molecules within the hydrophobic pocket, sphingosine release; the reverse VP2 2 axis parallel alpha the spiral like both sides of pan, formed more than 9 in diameter of the "hole", buried in the VP1 N 2 axis just below the end of the virus will migrate to the outside, in nucleic acid release channel. So screening for biological mechanism of different binding epitope of the monoclonal antibody is helpful to further elaborate the virus uncoating. The subject of inactivated virus and recombinant capsid protein Balb/C mice were immunized using classical hybridoma screening technique, obtained 15 strains of monoclonal antibody could bind to the EV71 virus, including 6 strains of neutralizing antibody of animal protection. Experiments show that in the dose of 10ug/g rat weight antibody, protection efficiency of 6G5,12B6,2B6,2G12 can reach 100%, the antibody dose of 2ug/g, 6G5 and the protection rate could still maintain 100%, 12B6 can achieve 80% protection, higher than the current literature and protective monoclonal antibody. The EV71 can cause sterility meningitis, encephalitis (Asepic meningitis) (encephalitis), encephalomyelitis (encephalomyelitis) and acute flaccid paralysis (acute flaccid, paralysis, AFP) and severe neurological complications, and protective effect on nerve cells we also tested 15 strains of monoclonal antibodies, compared with human rhabdomyoma cells in the same titer of virus attacks, the protective effect of 2B6 has been greatly improved. The reason is that because the EV71 resulted in the invasion mechanism of muscle tissue and the nervous system is not the same. According to the in vivo protection function and the similarities and differences between epitopes can be divided into the study and protection of antibody of 3 kinds, the first kind of 2B6,2G12 can be combined with VP1-GHloop 215-KQEKD-219, second 7G6,12H3 VP1 N recognition terminal 4-VADVI-8 third class 6G5,12B6 space for the identification of epitopes, 6G5 epitopes, we use electron cryomicroscopy composite structure, analysis of 6G5Fab and three different kinds of viruses of the menstrual cycle, including 6G5Fab- natural mature virus, 6G5Fab- virus and 6G5Fab- virus hollow hollow shrunken, the resolution reached 5.6, between 4.9 and 5.6G5 recognition sites located on viral particles 2 axis and 3 axis, at the intersection of VP2 distribution and VP3, including VP2 135-139 of the LDTKL, 223-226 GADG, 296-298 GAT and VP3 73-81 a bit VSAQAGKGE, 144-147 KDRA, 209-212 PNTA. which consists of a groove"" Handle complex binding region "and" Canyon "composition. The key amino acid epitope interaction with virus by mutation of 6G5 variable region on the combination of the 6 functional domains found the spatial epitopes were involved in 6G5 and virus. And then we deeply study the protective mechanism of three kinds of antibodies, found structure the combination of 2B6 by reducing the flexibility of VP1-GHloop to stabilize near 5 axis at the bottom of the canyon, conformational changes which inhibit the virus, the virus removal effect of particle formation process; 7G6 can block the viral nucleic acid channel, inhibiting viral nucleic acid release; the most interesting is the protective mechanism of 6G5, it can deceive the virus. Play a" receptor "function, uncoating process advance mediated cell, induced by the viral genome and inhibition of 135S release, the formation of uncoating intermediates. The above results we believe that the combination of 6G5 based on The site is a new neutralizing epitope. Its protection mechanism has brought a whole new train of thought for the strategy of antiviral therapy. This research achievement is also helpful to understand the neutralization protection mechanism of anti EV71 virus infection, and lay a solid foundation for the development of antibody drugs.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

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本文編號(hào)：1453744