埃博拉病毒等高致病性传染病假病毒小鼠感染模型的建立及应用
发布时间：2018-07-28 13:19
【摘要】：新发突发传染病和烈性传染病病原体是目前公共卫生领域研究的热点和难点。2014年暴发的埃博拉病毒(EBOV)出血热,在几内亚、利比里亚和塞拉利昂等西非国家广泛传播,引起了全世界的关注。与EBOV同属丝状病毒科的马尔堡病毒(MARV),致死率高达90%。2012年新发传染病中东呼吸综合征(MERS),是由一种新型冠状病毒(MERS-CoV)引起的病毒性呼吸道疾病,已在包括我国在内的多个国家发现输入病例。拉沙热病毒(LASV)主要经啮齿类动物传播给人,具有大规模传播的风险,其预防和治疗至关重要。目前,上述病毒的病原学、致病机制等尚未完全认识清楚,且研究上述病毒所需实验室的生物安全等级要求高,缺少经济有效的动物模型等因素,限制了针对此类病毒的药物和疫苗研发。为降低高致病性病毒的生物风险,可通过构建假病毒的方法研究烈性病毒。因此,本课题旨在通过构建高滴度假病毒和假病毒感染小鼠活体成像模型,以解决上述瓶颈问题。本课题组前期已经对假病毒包装体系中的骨架质粒pSG3Δenv进行改造,构建出了携带萤火虫荧光素酶(Fluc)基因的骨架质粒,提高了假病毒的滴度。本课题优化了假病毒包装体系的影响因素:包装细胞、表达质粒、骨架质粒、表达质粒与骨架质粒的比例、转染试剂,从而将EBOV、MARV、MERS-CoV、LASV假病毒滴度提高了100~1000倍。本课题利用非复制型假病毒创新性建立了一系列安全、高通量的抗病毒活性检测方法,并进行影响因素标准化研究,包括细胞嗜性、检测时间、细胞接种量、病毒加入量等。此系列方法灵敏度高、特异性、重复性好,可用于疫苗、单抗和小分子化合物等抗病毒制品的高通量筛选和效力评价。本课题在EBOV、MARV、MERS-CoV、LASV高滴度假病毒的基础上,通过优化假病毒感染小鼠的品系、周龄、感染途径、检测时间等,首次建立了EBOV、MARV、MERS-CoV、LASV假病毒可视化小鼠感染模型,可动态观察假病毒感染小鼠后的体内分布和发光强度,用于疫苗、抗体、抗病毒抑制剂的体内保护效果评价。本课题制备的EBOV假病毒滴度可达1×107TCID50/m L,敏感细胞株为293T,在体内外对义翘神州公司提供的四株鼠源性单抗的中和活性进行评价,结果显示,M318和M501中和活性强。MARV假病毒滴度可达9.75×107TCID50/m L,敏感细胞株为293T,使用假病毒免疫豚鼠得到的血清具有中和活性,在小鼠感染模型中,血清具有很好的被动免疫保护效果。MERS-CoV假病毒滴度可达1.27×108TCID50/mL,敏感细胞株为Huh 7,小鼠模型为DPP4 KI模式动物,在体内外水平验证了义翘神州公司提供的单抗H111-1和R723-NEU具有强中和活性。LASV假病毒滴度可达1.67×108TCID50/mL,敏感细胞株为293T,使用DNA疫苗和假病毒颗粒疫苗免疫小鼠,表明疫苗具有很好的保护性。本课题成功构建了一系列基于假病毒的安全、灵敏且特异性的抗病毒活性细胞学评价方法,以及可视化的假病毒小鼠感染模型。此检测平台可在BSL-2实验室操作,解决了高致病性传染病病原学研究及相关制品效力评价的技术瓶颈,对于药物、疫苗、小分子化合物等抗病毒制品的抗病毒活性高通量筛选和效力评价提供了理想的检测技术平台。
[Abstract]:The new outbreak of infectious diseases and infectious diseases is a hot and difficult problem in the field of public health research at present. The outbreak of Ebola virus (EBOV) hemorrhagic fever (.2014) is widely spread in Guinea, Liberia and Sierra Leone and other West African countries. It has attracted worldwide attention. EBOV, which belongs to the family of filiform virus (MARV), is the same as that of the family of filamentous viruses. 90%.2012, a new infectious disease of the Middle East respiratory syndrome (MERS), is a viral respiratory disease caused by a new type of coronavirus (MERS-CoV), which has been found in many countries, including our country. The Lassa fever virus (LASV) is transmitted mainly by rodents, with the risk of mass transmission, and the prevention and prevention of the disease. Treatment is of vital importance. At present, the etiology of the virus, the pathogenesis, and so on are not fully understood, and the biological safety level of the laboratory is high, the lack of effective animal models and other factors, limiting the development of the drugs and vaccine against the virus, and the biological risk of reducing the high pathogenic virus, The aim of this study is to solve the problem of the above bottleneck by constructing a high drop holiday virus and a pseudo virus infected mouse living imaging model. In the earlier period, the skeleton plasmid pSG3 delta env in the pseudo virus packaging system was reformed and the firefly luciferase was constructed. (Fluc) the skeleton plasmid of the gene improved the titer of the false virus. This topic optimized the influencing factors of the pseudo virus packaging system: the proportion of the packaging cells, the expression plasmids, the skeleton plasmids, the expression plasmid and the skeleton plasmid, the transfection reagent, so that the titer of the EBOV, MARV, MERS-CoV and LASV pseudo virus increased by 100~1000 times. The virus innovatively established a series of safety, high throughput antiviral activity detection methods and standardized research on influence factors, including cell tropism, detection time, cell inoculation, virus addition, etc. This series of methods have high sensitivity, specificity and reproducibility, and can be used in antiviral products such as vaccines, monoclonal antibodies and small molecular compounds. High throughput screening and effectiveness evaluation. On the basis of EBOV, MARV, MERS-CoV, LASV high drop holiday virus, we first established a model of EBOV, MARV, MERS-CoV, LASV pseudo virus infection in mice by optimizing the strain of the pseudo virus infection in mice, the way of infection, and the detection time. It can dynamically observe the body of the mice infected by the false virus. Distribution and luminescence intensity were used to evaluate the protective effect of vaccines, antibodies, and antiviral inhibitors in vivo. The EBOV pseudo virus titer prepared by this study was 1 * 107TCID50/m L, and the sensitive cell strain was 293T. The neutralization activity of four mouse derived McAbs was evaluated in vivo and in vitro. The results showed that the neutralization activity of M318 and M501 was found. The strong.MARV pseudo virus titer can reach 9.75 * 107TCID50/m L, the sensitive cell line is 293T, the sera obtained by the pseudo virus immunized guinea pig has neutralization activity. In the mouse infection model, the serum has a good passive immune protection effect,.MERS-CoV pseudo virus titer can reach 1.27 x 108TCID50/mL, the sensitive cell line is Huh 7, and the mouse model is DPP4 KI The model animals, in vivo and in vitro, demonstrated that the strong neutralizing active.LASV pseudo virus titer of H111-1 and R723-NEU was 1.67 * 108TCID50/mL, the sensitive cell strain was 293T, the DNA vaccine and the pseudo virus particle vaccine were used to immunize mice, which showed that the vaccine had good protection. Safety, sensitive and specific antiviral activity cytology evaluation method based on false virus, and visual pseudo virus mouse infection model. This detection platform can be operated in the BSL-2 laboratory. It solves the technical bottleneck of the pathogenic study of highly pathogenic infectious diseases and the effectiveness evaluation of related products, and the combination of drugs, vaccines and small molecules. High throughput screening and potency evaluation of antiviral activity such as antiviral products provide an ideal platform for detection.
【学位授予单位】：中国食品药品检定研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.8;R-332
本文编号：2150279
[Abstract]:The new outbreak of infectious diseases and infectious diseases is a hot and difficult problem in the field of public health research at present. The outbreak of Ebola virus (EBOV) hemorrhagic fever (.2014) is widely spread in Guinea, Liberia and Sierra Leone and other West African countries. It has attracted worldwide attention. EBOV, which belongs to the family of filiform virus (MARV), is the same as that of the family of filamentous viruses. 90%.2012, a new infectious disease of the Middle East respiratory syndrome (MERS), is a viral respiratory disease caused by a new type of coronavirus (MERS-CoV), which has been found in many countries, including our country. The Lassa fever virus (LASV) is transmitted mainly by rodents, with the risk of mass transmission, and the prevention and prevention of the disease. Treatment is of vital importance. At present, the etiology of the virus, the pathogenesis, and so on are not fully understood, and the biological safety level of the laboratory is high, the lack of effective animal models and other factors, limiting the development of the drugs and vaccine against the virus, and the biological risk of reducing the high pathogenic virus, The aim of this study is to solve the problem of the above bottleneck by constructing a high drop holiday virus and a pseudo virus infected mouse living imaging model. In the earlier period, the skeleton plasmid pSG3 delta env in the pseudo virus packaging system was reformed and the firefly luciferase was constructed. (Fluc) the skeleton plasmid of the gene improved the titer of the false virus. This topic optimized the influencing factors of the pseudo virus packaging system: the proportion of the packaging cells, the expression plasmids, the skeleton plasmids, the expression plasmid and the skeleton plasmid, the transfection reagent, so that the titer of the EBOV, MARV, MERS-CoV and LASV pseudo virus increased by 100~1000 times. The virus innovatively established a series of safety, high throughput antiviral activity detection methods and standardized research on influence factors, including cell tropism, detection time, cell inoculation, virus addition, etc. This series of methods have high sensitivity, specificity and reproducibility, and can be used in antiviral products such as vaccines, monoclonal antibodies and small molecular compounds. High throughput screening and effectiveness evaluation. On the basis of EBOV, MARV, MERS-CoV, LASV high drop holiday virus, we first established a model of EBOV, MARV, MERS-CoV, LASV pseudo virus infection in mice by optimizing the strain of the pseudo virus infection in mice, the way of infection, and the detection time. It can dynamically observe the body of the mice infected by the false virus. Distribution and luminescence intensity were used to evaluate the protective effect of vaccines, antibodies, and antiviral inhibitors in vivo. The EBOV pseudo virus titer prepared by this study was 1 * 107TCID50/m L, and the sensitive cell strain was 293T. The neutralization activity of four mouse derived McAbs was evaluated in vivo and in vitro. The results showed that the neutralization activity of M318 and M501 was found. The strong.MARV pseudo virus titer can reach 9.75 * 107TCID50/m L, the sensitive cell line is 293T, the sera obtained by the pseudo virus immunized guinea pig has neutralization activity. In the mouse infection model, the serum has a good passive immune protection effect,.MERS-CoV pseudo virus titer can reach 1.27 x 108TCID50/mL, the sensitive cell line is Huh 7, and the mouse model is DPP4 KI The model animals, in vivo and in vitro, demonstrated that the strong neutralizing active.LASV pseudo virus titer of H111-1 and R723-NEU was 1.67 * 108TCID50/mL, the sensitive cell strain was 293T, the DNA vaccine and the pseudo virus particle vaccine were used to immunize mice, which showed that the vaccine had good protection. Safety, sensitive and specific antiviral activity cytology evaluation method based on false virus, and visual pseudo virus mouse infection model. This detection platform can be operated in the BSL-2 laboratory. It solves the technical bottleneck of the pathogenic study of highly pathogenic infectious diseases and the effectiveness evaluation of related products, and the combination of drugs, vaccines and small molecules. High throughput screening and potency evaluation of antiviral activity such as antiviral products provide an ideal platform for detection.
【学位授予单位】：中国食品药品检定研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.8;R-332
【参考文献】
相关期刊论文 前1条
1 程颖;刘军;李昱;刘翟;任翔;施一;高福;余宏杰;;埃博拉病毒病:病原学、致病机制、治疗与疫苗研究进展[J];科学通报;2014年30期
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