中东呼吸综合征冠状病毒病毒样颗粒的构建与应用研究

发布时间：2018-06-20 12:34

本文选题：中东呼吸综合征 + 冠状病毒　；参考：《东北林业大学》2016年博士论文

【摘要】：中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)于2012年首次分离,是一种可感染人类并致死的新型冠状病毒。中东呼吸综合征由MERS-CoV感染所引起,症状表现包括发热、咳嗽、急性呼吸系统窘迫征等,严重者死亡,病死率达35%以上。MERS-CoV已在27个国家造成感染,感染病例多发生于中东地区,但在非洲,亚洲,南美,均有感染病例,且均与中东地区有关联。随着世界经济全球化,我国与其它国家联系紧密,往来频繁,增加了自然感染或输入性病例的几率。目前,对于MERS尚无获批的预防性疫苗或特异性治疗方法。研制用于MERS预防和治疗的疫苗和治疗性抗体对于该病的防控、保护患者与医疗工作者的生命安全具有重要意义。2015年在广州有一起由韩国输入的MERS病例,对我国的MERS疫情防控敲响警钟。中国人口密度大,为防止“非典”疫情重演,研制安全性高,免疫原性强的疫苗及快速救治药物,用于MERS防控储备,具有重要意义。MERS-CoV生物安全风险高,操作活病毒可能带来生物安全问题,需要在生物安全等级三级实验室进行,且需要专业人员操作,传统的灭活疫苗和弱毒疫苗研究及其规模化生产遇到障碍,因此探索安全有效的免疫原对于候选疫苗的制备与使用具有重要的现实意义。病毒样颗粒(virus-like particles, VLPs)是一种由表达的病毒结构蛋白自发装配而成,形似真实病毒的颗粒；其本质为蛋白质,不含病毒核酸,无感染或复制能力,安全性高；VLPs由一个或多个蛋白亚单位构成,由于其呈高密度、重复排列,使其易于被免疫系统识别,因而具有良好的免疫原性。为构建MERS-CoV VLPs,将MERS-CoV纤突蛋白(Spike, S)、膜蛋白(membrane, M)和包膜蛋白(envelope protein,E)基因重组至杆状病毒表达载体,拯救重组杆状病毒。经基因组PCR与间接免疫荧光等方法鉴定,获得共表达S、M和E基因的重组杆状病毒。将重组杆状病毒感染Sf9昆虫细胞,收获细胞上清液并通过蔗糖梯度密度离心纯化后,电镜观察到形状类似冠状病毒的病毒样颗粒,经免疫电镜与Western blot方法鉴定,获得MERS-CoV VLPs。为评价MERS-CoV VLPs的免疫原性,以纯化的MERS-CoV VLPs作为免疫原,免疫BALB/c鼠和恒河猴,进行MERS-CoV VLPs免疫原性的评价。通过酶联免疫吸附实验测定小鼠和恒河猴血清IgG,平均效价分别为1：384和1：1067;经中和实验测定小鼠和恒河猴血清中和抗体,平均效价分别为1：208和1：33。通过酶联免疫斑点实验对免疫动物细胞因子IFN-γ、IL-4的分泌情况进行评价,结果显示小鼠经MERS-CoV VLPs免疫后,在特异性抗原刺激下,可分泌IL-4显著提高,而恒河猴分泌IFN-γ显著增多。上述结果表明MERS-CoV VLPs对小鼠和恒河猴具有免疫原性,经免疫的小鼠和恒河猴可诱导产生特异性抗体反应与细胞免疫反应。因此,IERS-CoV VLPs具有开发成为安全有效的新型中东呼吸综合征疫苗的潜能。抗血清疗法是治疗病毒和细菌等感染的快速、经济、有效手段。因此,本研究将MERS-CoV VLPs作为免疫原对健康马匹进行多次免疫,获得马抗MERS-CoV高免血清。通过中和试验监测马血清抗体效价,结果表明抗体效价与免疫次数呈正相关,在5次免疫后,马抗MERS-CoV高免血清中和活性为1：20900；在MERS-CoV小鼠感染模型上攻毒保护试验,分别攻毒前后肌肉注射马抗MERS-CoV高免血清可以显著降低攻毒小鼠肺内病毒滴度,证明其具有预防及暴露后的保护作用。马抗MERS-CoV高免血清具备有效性,为MERS紧急救治药物研究奠定了基础。为进一步探索MERS-CoV受体结合区(receptor-binding domain,RBD)作为亚单位疫苗的可行性,利用VLPs具有展示外源表位的特性,利用表达单个蛋白即可形成病毒样颗粒的犬细小病毒(canine parvovirus,CPV) VP2蛋白作为载体,融合表达MERS-CoV RBD基因。通过对重组杆状病毒提取基因组进行目的片段特异性扩增与间接免疫荧光试验等方法进行鉴定,获得融合表达MERS-CoV RBD和CPV VP2基因的重组杆状病毒。将重组杆状病毒感染Sf9昆虫细胞,收获细胞并利用饱和硫酸铵法纯化VLPs,在电镜下可观察到形状类似细小病毒的VLPs。通过免疫电镜与Western blot方法对VLPs进行鉴定,结果表明获得将MERS-CoV RBD蛋白展示于表面的嵌合型VLPs。为评价嵌合型MERS-CoV VLPs的免疫原性,将其作为免疫原,在BALB/c鼠上进行免疫原性的评价。通过酶联免疫吸附试验测定小鼠血清中IgG抗体效价为1：416；通过假病毒中和实验测定小鼠血清的中和抗体效价为1：72。分离小鼠脾细胞并进行酶联免疫斑点试验,发现小鼠经免疫后在特异性抗原刺激下可分泌显著增多的细胞因子IFN-γ、IL-4和IL-2。分离小鼠腹股沟淋巴结中的淋巴细胞并进行抗体标记后通过流式细胞检测,发现免疫嵌合型VLPs对树突细胞具有募集活化的能力。上述结果说明,经嵌合型VLPs免疫的小鼠可诱导产生特异性抗体反应与细胞免疫反应。结果提示MERS-CoV嵌合型VLPs具有开发成为MERS疫苗候选株的潜能。本研究立足于MERS-CoV候选疫苗与抗体的初步研究,构建制备MERS-CoV疫苗候选株和马高免血清,分析MERS VLPs的结构特点与免疫机制,为防控MERS疫情及其它外来人兽共患病提供了技术储备。
[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.
【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R373

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