尼帕脑炎病毒G和F蛋白DNA疫苗对小鼠免疫原性的研究

发布时间：2018-01-31 01:17

本文关键词： 尼帕脑炎病毒 G蛋白 F蛋白 DNA疫苗体液免疫细胞免疫　出处：《中国农业科学院》2012年硕士论文　论文类型：学位论文

【摘要】：尼帕病脑炎是由尼帕病毒（Nipah Virus，NiV）引起的一种烈性人畜共患病。1999年以来，马来西亚、新加坡、孟加拉国、印度等东南亚、南亚国家先后爆发尼帕病脑炎，，人类感染死亡率达40％以上。NiV属副粘病毒科亨尼帕病毒属，其自然宿主为果蝠，在东南亚及我国南方地区广泛分布。近年，我国南方部分地区的果蝠体内已检测到尼帕病毒抗体的存在，提示尼帕病毒已对我国的公共卫生构成现实的威胁。目前，尼帕病脑炎尚无有效治疗方法，研制安全有效的疫苗，具有重要意义。 DNA疫苗能够刺激机体产生较全面的免疫反应，和灭活疫苗、减毒疫苗、亚单位疫苗相比，具有独特的优势。NiV的囊膜糖蛋白G和F是诱导中和抗体的主要免疫原。本研究利用分别表达G和F蛋白的真核表达质粒pCAGG-NiVG和pCAGG-NiVF单独或联合免疫BALB/c小鼠两次；免疫小鼠的体液免疫反应采用酶联免疫吸附试验（ELISA）和水泡性口炎病毒嵌合NiV囊膜糖蛋白G/F蛋白伪型病毒粒子（VSV△G*F/G）分别检测小鼠血清中NiV特异性IgG抗体和中和抗体水平；为了检测免疫小鼠的细胞免疫水平，本研究利用NiV F蛋白CD8+T细胞表位（VYFPILTEI,H2Kd）多肽为刺激物采用流式细胞术（FCM）和酶联免疫斑点实验（ELISPOT）检测pCAGG-NiVF免疫小鼠的的特异性CD8+T细胞免疫反应。本研究结果显示pCAGG-NiVG和pCAGG-NiVF单独免疫小鼠，初次免疫后即可诱导产生NiV特异性IgG抗体和中和抗体；加强免疫后这两种抗体水平显著上升（p0.05）。pCAGG-NiVG和pCAGG-NiVF诱导相似水平的ELISA抗体和中和抗体，联合免疫组与单独免疫组小鼠相比体液免疫水平没有显著区别（p0.05）。FCM和ELISPOT检测结果显示pCAGG-NiVF初次免疫小鼠后即能够产生良好的特异性CD8+T细胞免疫应答，且该细胞免疫反应在小鼠二次免疫后显著增强（p0.05）。表达NiV G和F蛋白的DNA疫苗能够诱导小鼠产生特异性体液免疫和细胞免疫反应，在小鼠上具有良好的免疫原性，是一种有希望的尼帕病脑炎候选疫苗。
[Abstract]:Nipa encephalitis is a severe zoonosis caused by Nipah virus. Since 1999, Malaysia, Singapore, Bangladesh, India and other Southeast Asia. Nipa disease encephalitis has been outbreak in South Asian countries, and the mortality rate of human infection is more than 40%. NiV belongs to the genus Cohenipa virus of paramyxovirus, and its natural host is fruit bat. It is widely distributed in Southeast Asia and southern China. In recent years, the presence of Nippa virus antibodies has been detected in fruit bats in parts of southern China. It is suggested that Nipavirus has posed a real threat to public health in China. At present, there is no effective treatment for Nipah's encephalitis, and it is of great significance to develop a safe and effective vaccine. DNA vaccine can stimulate the body to produce a more comprehensive immune response, compared with inactivated vaccine, attenuated vaccine, subunit vaccine. In this study, the eukaryotic expression plasmids pCAGG-NiVG and pCAG, which expressed G and F proteins respectively, were used to induce neutralizing antibodies. BALB/c mice were immunized with G-NiVF alone or in combination twice. Humoral immune response of immunized mice by enzyme linked immunosorbent assay (Elisa) and vesicular stomatitis virus chimeric NiV envelope glycoprotein G / F protein pseudoviral particle. The levels of NiV specific IgG antibody and neutralizing antibody in serum of mice were detected respectively. In order to detect the cellular immunity of immunized mice, the NiV F protein CD8 T cell epitope VYFPILTEI was used in this study. H2KD polypeptide was stimulated by flow cytometry (FCM) and enzyme-linked immunoblot assay (ELISPOT). The specific CD8 T cell immune response of mice immunized with pCAGG-NiVF was detected. The results showed that mice immunized with pCAGG-NiVG and pCAGG-NiVF alone could induce the production of NiV specific IgG antibody and neutralizing antibody after primary immunization. The levels of these two antibodies increased significantly after immunization. PCAGG-NiVG and pCAGG-NiVF induced similar levels of ELISA antibodies and neutralizing antibodies. There was no significant difference in humoral immunity level between the combined immunization group and the single immunization group (p 0.05). The results of FCM and ELISPOT showed that the mice immunized with pCAGG-NiVF for the first time could produce a good specific CD8 T cell immune response. The cellular immune reaction was significantly increased after the second immunization in mice. The DNA vaccine expressing NiV G and F proteins can induce specific humoral and cellular immune responses in mice and has good immunogenicity in mice. Is a promising candidate vaccine for Nippa's encephalitis.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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