埃博拉病毒等高致病性傳染病假病毒小鼠感染模型的建立及應(yīng)用
發(fā)布時間：2018-07-28 13:19
【摘要】：新發(fā)突發(fā)傳染病和烈性傳染病病原體是目前公共衛(wèi)生領(lǐng)域研究的熱點和難點。2014年暴發(fā)的埃博拉病毒(EBOV)出血熱,在幾內(nèi)亞、利比里亞和塞拉利昂等西非國家廣泛傳播,引起了全世界的關(guān)注。與EBOV同屬絲狀病毒科的馬爾堡病毒(MARV),致死率高達(dá)90%。2012年新發(fā)傳染病中東呼吸綜合征(MERS),是由一種新型冠狀病毒(MERS-CoV)引起的病毒性呼吸道疾病,已在包括我國在內(nèi)的多個國家發(fā)現(xiàn)輸入病例。拉沙熱病毒(LASV)主要經(jīng)嚙齒類動物傳播給人,具有大規(guī)模傳播的風(fēng)險,其預(yù)防和治療至關(guān)重要。目前,上述病毒的病原學(xué)、致病機(jī)制等尚未完全認(rèn)識清楚,且研究上述病毒所需實驗室的生物安全等級要求高,缺少經(jīng)濟(jì)有效的動物模型等因素,限制了針對此類病毒的藥物和疫苗研發(fā)。為降低高致病性病毒的生物風(fēng)險,可通過構(gòu)建假病毒的方法研究烈性病毒。因此,本課題旨在通過構(gòu)建高滴度假病毒和假病毒感染小鼠活體成像模型,以解決上述瓶頸問題。本課題組前期已經(jīng)對假病毒包裝體系中的骨架質(zhì)粒pSG3Δenv進(jìn)行改造,構(gòu)建出了攜帶螢火蟲熒光素酶(Fluc)基因的骨架質(zhì)粒,提高了假病毒的滴度。本課題優(yōu)化了假病毒包裝體系的影響因素:包裝細(xì)胞、表達(dá)質(zhì)粒、骨架質(zhì)粒、表達(dá)質(zhì)粒與骨架質(zhì)粒的比例、轉(zhuǎn)染試劑,從而將EBOV、MARV、MERS-CoV、LASV假病毒滴度提高了100~1000倍。本課題利用非復(fù)制型假病毒創(chuàng)新性建立了一系列安全、高通量的抗病毒活性檢測方法,并進(jìn)行影響因素標(biāo)準(zhǔn)化研究,包括細(xì)胞嗜性、檢測時間、細(xì)胞接種量、病毒加入量等。此系列方法靈敏度高、特異性、重復(fù)性好,可用于疫苗、單抗和小分子化合物等抗病毒制品的高通量篩選和效力評價。本課題在EBOV、MARV、MERS-CoV、LASV高滴度假病毒的基礎(chǔ)上,通過優(yōu)化假病毒感染小鼠的品系、周齡、感染途徑、檢測時間等,首次建立了EBOV、MARV、MERS-CoV、LASV假病毒可視化小鼠感染模型,可動態(tài)觀察假病毒感染小鼠后的體內(nèi)分布和發(fā)光強(qiáng)度,用于疫苗、抗體、抗病毒抑制劑的體內(nèi)保護(hù)效果評價。本課題制備的EBOV假病毒滴度可達(dá)1×107TCID50/m L,敏感細(xì)胞株為293T,在體內(nèi)外對義翹神州公司提供的四株鼠源性單抗的中和活性進(jìn)行評價,結(jié)果顯示,M318和M501中和活性強(qiáng)。MARV假病毒滴度可達(dá)9.75×107TCID50/m L,敏感細(xì)胞株為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,血清具有很好的被動免疫保護(hù)效果。MERS-CoV假病毒滴度可達(dá)1.27×108TCID50/mL,敏感細(xì)胞株為Huh 7,小鼠模型為DPP4 KI模式動物,在體內(nèi)外水平驗證了義翹神州公司提供的單抗H111-1和R723-NEU具有強(qiáng)中和活性。LASV假病毒滴度可達(dá)1.67×108TCID50/mL,敏感細(xì)胞株為293T,使用DNA疫苗和假病毒顆粒疫苗免疫小鼠,表明疫苗具有很好的保護(hù)性。本課題成功構(gòu)建了一系列基于假病毒的安全、靈敏且特異性的抗病毒活性細(xì)胞學(xué)評價方法,以及可視化的假病毒小鼠感染模型。此檢測平臺可在BSL-2實驗室操作,解決了高致病性傳染病病原學(xué)研究及相關(guān)制品效力評價的技術(shù)瓶頸,對于藥物、疫苗、小分子化合物等抗病毒制品的抗病毒活性高通量篩選和效力評價提供了理想的檢測技術(shù)平臺。
[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.
【學(xué)位授予單位】：中國食品藥品檢定研究院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.8;R-332
本文編號：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.
【學(xué)位授予單位】：中國食品藥品檢定研究院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.8;R-332
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 程穎;劉軍;李昱;劉翟;任翔;施一;高福;余宏杰;;埃博拉病毒病:病原學(xué)、致病機(jī)制、治療與疫苗研究進(jìn)展[J];科學(xué)通報;2014年30期
，本文編號：2150279
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