本文選題：中東呼吸綜合征 + 冠狀病毒��； 參考：《東北林業(yè)大學(xué)》2016年博士論文

【摘要】：中東呼吸綜合征冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)于2012年首次分離,是一種可感染人類并致死的新型冠狀病毒。中東呼吸綜合征由MERS-CoV感染所引起,癥狀表現(xiàn)包括發(fā)熱、咳嗽、急性呼吸系統(tǒng)窘迫征等,嚴(yán)重者死亡,病死率達(dá)35%以上。MERS-CoV已在27個(gè)國(guó)家造成感染,感染病例多發(fā)生于中東地區(qū),但在非洲,亞洲,南美,均有感染病例,且均與中東地區(qū)有關(guān)聯(lián)。隨著世界經(jīng)濟(jì)全球化,我國(guó)與其它國(guó)家聯(lián)系緊密,往來(lái)頻繁,增加了自然感染或輸入性病例的幾率。目前,對(duì)于MERS尚無(wú)獲批的預(yù)防性疫苗或特異性治療方法。研制用于MERS預(yù)防和治療的疫苗和治療性抗體對(duì)于該病的防控、保護(hù)患者與醫(yī)療工作者的生命安全具有重要意義。2015年在廣州有一起由韓國(guó)輸入的MERS病例,對(duì)我國(guó)的MERS疫情防控敲響警鐘。中國(guó)人口密度大,為防止“非典”疫情重演,研制安全性高,免疫原性強(qiáng)的疫苗及快速救治藥物,用于MERS防控儲(chǔ)備,具有重要意義。MERS-CoV生物安全風(fēng)險(xiǎn)高,操作活病毒可能帶來(lái)生物安全問(wèn)題,需要在生物安全等級(jí)三級(jí)實(shí)驗(yàn)室進(jìn)行,且需要專業(yè)人員操作,傳統(tǒng)的滅活疫苗和弱毒疫苗研究及其規(guī)�；a(chǎn)遇到障礙,因此探索安全有效的免疫原對(duì)于候選疫苗的制備與使用具有重要的現(xiàn)實(shí)意義。病毒樣顆粒(virus-like particles, VLPs)是一種由表達(dá)的病毒結(jié)構(gòu)蛋白自發(fā)裝配而成,形似真實(shí)病毒的顆粒；其本質(zhì)為蛋白質(zhì),不含病毒核酸,無(wú)感染或復(fù)制能力,安全性高；VLPs由一個(gè)或多個(gè)蛋白亞單位構(gòu)成,由于其呈高密度、重復(fù)排列,使其易于被免疫系統(tǒng)識(shí)別,因而具有良好的免疫原性。為構(gòu)建MERS-CoV VLPs,將MERS-CoV纖突蛋白(Spike, S)、膜蛋白(membrane, M)和包膜蛋白(envelope protein,E)基因重組至桿狀病毒表達(dá)載體,拯救重組桿狀病毒。經(jīng)基因組PCR與間接免疫熒光等方法鑒定,獲得共表達(dá)S、M和E基因的重組桿狀病毒。將重組桿狀病毒感染Sf9昆蟲(chóng)細(xì)胞,收獲細(xì)胞上清液并通過(guò)蔗糖梯度密度離心純化后,電鏡觀察到形狀類似冠狀病毒的病毒樣顆粒,經(jīng)免疫電鏡與Western blot方法鑒定,獲得MERS-CoV VLPs。為評(píng)價(jià)MERS-CoV VLPs的免疫原性,以純化的MERS-CoV VLPs作為免疫原,免疫BALB/c鼠和恒河猴,進(jìn)行MERS-CoV VLPs免疫原性的評(píng)價(jià)。通過(guò)酶聯(lián)免疫吸附實(shí)驗(yàn)測(cè)定小鼠和恒河猴血清IgG,平均效價(jià)分別為1：384和1：1067;經(jīng)中和實(shí)驗(yàn)測(cè)定小鼠和恒河猴血清中和抗體,平均效價(jià)分別為1：208和1：33。通過(guò)酶聯(lián)免疫斑點(diǎn)實(shí)驗(yàn)對(duì)免疫動(dòng)物細(xì)胞因子IFN-γ、IL-4的分泌情況進(jìn)行評(píng)價(jià),結(jié)果顯示小鼠經(jīng)MERS-CoV VLPs免疫后,在特異性抗原刺激下,可分泌IL-4顯著提高,而恒河猴分泌IFN-γ顯著增多。上述結(jié)果表明MERS-CoV VLPs對(duì)小鼠和恒河猴具有免疫原性,經(jīng)免疫的小鼠和恒河猴可誘導(dǎo)產(chǎn)生特異性抗體反應(yīng)與細(xì)胞免疫反應(yīng)。因此,IERS-CoV VLPs具有開(kāi)發(fā)成為安全有效的新型中東呼吸綜合征疫苗的潛能�？寡瀵煼ㄊ侵委煵《竞图�(xì)菌等感染的快速、經(jīng)濟(jì)、有效手段。因此,本研究將MERS-CoV VLPs作為免疫原對(duì)健康馬匹進(jìn)行多次免疫,獲得馬抗MERS-CoV高免血清。通過(guò)中和試驗(yàn)監(jiān)測(cè)馬血清抗體效價(jià),結(jié)果表明抗體效價(jià)與免疫次數(shù)呈正相關(guān),在5次免疫后,馬抗MERS-CoV高免血清中和活性為1：20900；在MERS-CoV小鼠感染模型上攻毒保護(hù)試驗(yàn),分別攻毒前后肌肉注射馬抗MERS-CoV高免血清可以顯著降低攻毒小鼠肺內(nèi)病毒滴度,證明其具有預(yù)防及暴露后的保護(hù)作用。馬抗MERS-CoV高免血清具備有效性,為MERS緊急救治藥物研究奠定了基礎(chǔ)。為進(jìn)一步探索MERS-CoV受體結(jié)合區(qū)(receptor-binding domain,RBD)作為亞單位疫苗的可行性,利用VLPs具有展示外源表位的特性,利用表達(dá)單個(gè)蛋白即可形成病毒樣顆粒的犬細(xì)小病毒(canine parvovirus,CPV) VP2蛋白作為載體,融合表達(dá)MERS-CoV RBD基因。通過(guò)對(duì)重組桿狀病毒提取基因組進(jìn)行目的片段特異性擴(kuò)增與間接免疫熒光試驗(yàn)等方法進(jìn)行鑒定,獲得融合表達(dá)MERS-CoV RBD和CPV VP2基因的重組桿狀病毒。將重組桿狀病毒感染Sf9昆蟲(chóng)細(xì)胞,收獲細(xì)胞并利用飽和硫酸銨法純化VLPs,在電鏡下可觀察到形狀類似細(xì)小病毒的VLPs。通過(guò)免疫電鏡與Western blot方法對(duì)VLPs進(jìn)行鑒定,結(jié)果表明獲得將MERS-CoV RBD蛋白展示于表面的嵌合型VLPs。為評(píng)價(jià)嵌合型MERS-CoV VLPs的免疫原性,將其作為免疫原,在BALB/c鼠上進(jìn)行免疫原性的評(píng)價(jià)。通過(guò)酶聯(lián)免疫吸附試驗(yàn)測(cè)定小鼠血清中IgG抗體效價(jià)為1：416；通過(guò)假病毒中和實(shí)驗(yàn)測(cè)定小鼠血清的中和抗體效價(jià)為1：72。分離小鼠脾細(xì)胞并進(jìn)行酶聯(lián)免疫斑點(diǎn)試驗(yàn),發(fā)現(xiàn)小鼠經(jīng)免疫后在特異性抗原刺激下可分泌顯著增多的細(xì)胞因子IFN-γ、IL-4和IL-2。分離小鼠腹股溝淋巴結(jié)中的淋巴細(xì)胞并進(jìn)行抗體標(biāo)記后通過(guò)流式細(xì)胞檢測(cè),發(fā)現(xiàn)免疫嵌合型VLPs對(duì)樹(shù)突細(xì)胞具有募集活化的能力。上述結(jié)果說(shuō)明,經(jīng)嵌合型VLPs免疫的小鼠可誘導(dǎo)產(chǎn)生特異性抗體反應(yīng)與細(xì)胞免疫反應(yīng)。結(jié)果提示MERS-CoV嵌合型VLPs具有開(kāi)發(fā)成為MERS疫苗候選株的潛能。本研究立足于MERS-CoV候選疫苗與抗體的初步研究,構(gòu)建制備MERS-CoV疫苗候選株和馬高免血清,分析MERS VLPs的結(jié)構(gòu)特點(diǎn)與免疫機(jī)制,為防控MERS疫情及其它外來(lái)人獸共患病提供了技術(shù)儲(chǔ)備。
[Abstract]:The Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV) was first isolated in 2012. It is a new type of coronavirus that can infect human and death. The Middle East respiratory syndrome is caused by MERS-CoV infection. The symptoms include fever, cough, acute respiratory distress syndrome, serious death and disease. The death rate of more than 35%.MERS-CoV has caused infection in 27 countries. Infection cases are mostly born in the Middle East, but in Africa, Asia and South America, all cases are infected and are related to the Middle East. With the globalization of the world economy, our country is closely connected with other countries and is frequent, increasing the probability of natural infection or inputting cases. Before, there is no approved preventive vaccine or specific treatment for MERS. Development of vaccines and therapeutic antibodies for MERS prevention and treatment, protection of the disease, protection of patients and medical workers' life safety are of great significance in Guangzhou, in Guangzhou, there was a case of MERS, which was lost in Korea by Korea, and the prevention and control of the MERS epidemic in China. China has a large population density. In order to prevent the recurrence of SARS epidemic, the development of high safety, immunogenicity vaccine and rapid treatment drugs for MERS prevention and control reserve is of great significance to the high biosafety risk of.MERS-CoV, the operation of live virus may bring about biological safety problems, and it needs to be carried out in the three level laboratory of biological safety grade. It is necessary for professional personnel to operate the traditional inactivated vaccine and attenuated vaccine and its large-scale production encountered obstacles. Therefore, it is of great practical significance to explore the safe and effective immunogen for the preparation and use of candidate vaccines. Virus-like particles (VLPs) is a kind of spontaneous assembly of expressed viral structural proteins. It is a particle like a real virus; its essence is protein, no virus nucleic acid, no infection or replication, high safety; VLPs is made up of one or more protein subunits. Because of its high density, repeated arrangement makes it easy to be identified by the immune system and thus has good immunogenicity. To construct MERS-CoV VLPs, MERS-CoV Spike (S), membrane protein (membrane, M) and envelope protein (E) genes were reorganized to baculovirus expression vector to save recombinant baculovirus. The recombinant baculovirus, which co expressed S, M and E genes, was obtained by genomic PCR and indirect immunofluorescence. The recombinant baculovirus was infected by the recombinant baculovirus, and the harvested baculovirus was harvested. After purification of the supernatant and centrifuging through the sucrose gradient density, the virus like particles like coronavirus were observed by electron microscopy. The immunogenicity of MERS-CoV VLPs. was evaluated by the immunoelectron microscopy and Western blot method. The purified MERS-CoV VLPs was used as the immunogen, the BALB/c mice and Ganges RIver monkeys were immunized and the MERS-CoV V was carried out. The evaluation of LPs immunogenicity. The serum IgG of mice and Ganges RIver monkeys was measured by enzyme linked immunosorbent assay. The average titers were 1:384 and 1:1067 respectively. The neutralization test was used to determine the neutralization antibodies in mice and Ganges RIver monkeys. The average titer was 1:208 and 1:33., respectively, by the enzyme linked immunosorbent assay for the immune animal cytokines, IFN- gamma, IL-4. The results showed that the secretion of IL-4 in mice was significantly increased with specific antigen stimulated by MERS-CoV VLPs immunization, and the secretion of IFN- gamma in Ganges RIver monkeys was significantly increased. The results showed that MERS-CoV VLPs had immunogenicity to mice and Ganges RIver monkeys, and the immunized mice and Ganges RIver monkeys could induce specific antibody response and to produce specific antibody responses. Therefore, IERS-CoV VLPs has the potential of developing a new and effective new Middle East respiratory syndrome vaccine. Antiserum therapy is a rapid, economical and effective method for the treatment of virus and bacteria infection. Therefore, this study uses MERS-CoV VLPs as immunogen to immunize healthy horses for many times and obtain the high MERS-CoV high of horse resistance. Serum antibody titer was monitored by neutralization test. The results showed that the antibody titer was positively correlated with the number of immune responses. After 5 times of immunization, the high serum neutralization activity of mAb MERS-CoV was 1:20900, and in the MERS-CoV mice infection model, the anti serum MERS-CoV high serum free serum was significant before and after the attack. To reduce the virus titer in the lung of mice, it is proved that it has the protective effect after the prevention and exposure. The high serum free serum of Ma anti MERS-CoV is effective and lays the foundation for the study of MERS emergency treatment drugs. The feasibility of the MERS-CoV receptor binding region (receptor-binding domain, RBD) as a subunit vaccine is further explored, and VLPs is used in the development of VLPs. The characteristics of the exogenous epitopes are shown by using the canine parvovirus (CPV) VP2 protein, which can form a virus like particle, as a carrier, to express the MERS-CoV RBD gene, and to identify the genome of the recombinant baculovirus by specific expansion and indirect immunofluorescence test. Recombinant baculovirus expressing MERS-CoV RBD and CPV VP2 gene was fused. Recombinant baculovirus was infected with Sf9 insect cells, harvested cells were harvested and VLPs was purified by saturated ammonium sulfate method. Under electron microscope, VLPs. can be observed by immunoelectron microscopy and Western blot square method to identify VLPs. The results showed that MERS- was obtained. CoV RBD protein was displayed on the surface of chimeric type VLPs. to evaluate the immunogenicity of chimeric MERS-CoV VLPs. It was used as immunogen to evaluate immunogenicity on BALB/c mice. The titer of IgG antibody in serum of mice was determined by enzyme linked immunosorbent assay (ELISA), and the neutralization antibody of mouse serum was determined by the neutralization test of pseudo virus. The mice splenic cells were separated by 1:72. and the enzyme linked immunosorbent assay was carried out. It was found that the mice could secrete a significant increase of cytokine IFN- gamma after immunization with specific antigen, and IL-4 and IL-2. were used to separate the lymphocytes in the inguinal lymph nodes of mice and to detect the antibody by flow cytometry. The immune chimeric VLPs was found. Dendritic cells have the ability to raise activation. These results suggest that mice immunized with chimeric VLPs can induce specific antibody responses and cellular immune responses. The results suggest that the MERS-CoV chimeric VLPs has the potential to be a candidate for the development of a MERS vaccine. This study is based on the preliminary study of the MERS-CoV vaccine and the antibody. The MERS-CoV vaccine candidate and the horse high serum free serum were prepared, and the structural features and immune mechanisms of MERS VLPs were analyzed to provide technical reserves for the prevention and control of the MERS epidemic and other foreign zoonosis.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R373

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6 王，

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