淋巴细胞脉络丛脑膜炎病毒GP2单克隆抗体的制备和鉴定

发布时间：2018-09-15 20:07

【摘要】：研究背景淋巴细胞脉络丛脑膜炎病毒(LCMV)隶属于沙粒病毒科家族,自然宿主是啮齿类动物,常见的如家鼠、宠物鼠、仓鼠等都是它的天然携带者。它可以通过唾液、鼻分泌物、乳汁、精液、尿液和粪便使病毒得以传播,也可以通过其它途径,如皮肤、粘膜伤口、胎盘甚至病毒粒子气溶胶的形式传播。宿主感染了LCMV病毒后大都无明显的症状,这就成为了LCMV可以大面积地隐匿性传播的前提条件。人们很容易低估了LCMV感染的广泛性。通过对LCMV的研究,人类在诸如T细胞的MHC限制性、CD8+T细胞和CD4+T细胞在清除病毒过程中作用、T细胞耗竭、免疫耐受等方面取得了里程碑式的重大发现。其中的Armstrong和Cl-13两个亚型因为基因组的微小差异而引起宿主急慢性感染的巨大差异被众多实验室作为模式病毒,深入地研究免疫系统对急慢性感染的病理变化以及对慢性感染的深入研究。目前对LCMV慢性感染的认识也已经扩展到了HIV、结核病、乙肝、丙肝、自身免疫病和肿瘤的领域。而LCMV可以通过多种途径实现动物-人的传播。由于人类感染了LCMV病毒通常无明显症状,或者出现轻微的非特异性症状而自愈,故导致人们很容易忽视它的存在。而在特定条件下,如妊娠、免疫功能低下或者器官移植时出现机会感染,对人类健康造成极大威胁。GP2是LCMV穿膜蛋白,位于LCMV粒子表面,同GP1共同起修饰病毒表面的作用,同时它在病毒感染宿主细胞时担负了通过变构而触发病毒膜与细胞膜的融合功能。但目前国内外实验室对于LCMV的检测仍在使用免疫血清或者RT-PCR法,到目前为止暂未出现抗LCMV GP2的单克隆抗体的商品,因此研发抗LCMV GP2的mAb在科研和临床有重要的现实意义。研究目的本研究的目的是制备针对LCMV GP2的m Ab,为开发酶标或者荧光标记的检测抗体打下基础,为以后的免疫治疗提供可能;也可以利用该抗体亲和纯化GP2,从而进一步研究其结构和功能、LCMV疫苗的开发做好物质准备。实验方法采用杂交技术获得并筛选出杂交瘤细胞,收集含mAb的培养上清行免疫荧光(IFA)、dot-blot、ELISA法鉴定抗体重链类型和测定mAb的免疫效价。用蛋白G Sepharose亲和层析填料纯化抗体,纯化后的抗体采用SDS-PAGE检测纯化效果,并进一步用Western-blot检查抗体对LCMV的作用部位。实验结果成功获得能稳定分泌抗LCMV的特异性杂交瘤细胞株,顺利获得纯化的mAb。IFA、dot-blot结果均证实获得的mAb能识别LCMV抗原。用ELISA法鉴定出该mAb重链类型为IgG2b,培养液免疫效价约为1.35×103。蛋白G亲和层析填料纯化抗体后经SDS-PAGE考马斯亮蓝R250检测证实纯化抗体纯度高。Western-blot提示该m Ab对LCMV的作用部位为病毒颗粒表面糖蛋白抗原GP2。结论成功制备出了针对LCMV病毒颗粒表面糖蛋白抗原GP2高亲和力的m Ab,抗体类型是IgG2b。
[Abstract]:Background Lymphocytic choroid plexitis virus (LCMV) belongs to the family of the family Sagituliviridae, and its natural host is rodent, common such as domestic mouse, pet mouse, hamster and so on. It can be transmitted through saliva, nasal secretions, milk, semen, urine and faeces. It can also be transmitted in other ways, such as skin, mucosal wounds, placenta and even viral aerosols. Most of the hosts infected with LCMV virus have no obvious symptoms, which becomes the precondition for LCMV to be widely concealed. It is easy to underestimate the prevalence of LCMV infection. Through the study of LCMV, we have made a landmark discovery in such aspects as T cell MHC restricted CD8 T cells and CD4 T cells in the process of eliminating virus. The function of T cells is depletion of T cells, immune tolerance and so on. The two subtypes of Armstrong and Cl-13 have been used as model viruses in many laboratories because of the small differences in genomes that cause acute and chronic infection of the host. To study the pathological changes of the immune system in acute and chronic infection and the chronic infection. Knowledge of chronic LCMV infection has also expanded to include HIV, tuberculosis, hepatitis B, hepatitis C, autoimmune disease and cancer. And LCMV can achieve animal-human transmission through a variety of ways. It is easy to ignore the presence of LCMV virus in humans because it is usually asymptomatic or self-healing with mild nonspecific symptoms. Under certain conditions, such as pregnancy, low immune function or opportunistic infection during organ transplantation, GP2 is a great threat to human health. GP2 is a membrane penetrating protein of LCMV, which is located on the surface of LCMV particles and acts together with GP1 to modify the surface of virus. At the same time, it is responsible for the fusion function of virus membrane and cell membrane when the virus infects the host cell. But at present, the detection of LCMV in domestic and foreign laboratories is still using immune serum or RT-PCR method, so far, there are no monoclonal antibodies to LCMV GP2, so it is of great practical significance to develop anti-LCMV GP2 mAb in scientific research and clinic. Objective the purpose of this study was to prepare m Ab, for LCMV GP2 to lay a foundation for the development of enzyme-labeled or fluorescent labeled antibodies, and to provide the possibility for immunotherapy in the future. It can also be used to purify GP2, by affinity to further study its structure and function and to prepare for the development of LCMV vaccine. Methods hybridoma cells were obtained and screened by hybridization technique. The supernatants containing mAb were collected and immunofluorescence (IFA) dot-blot Elisa was used to identify the type of antibody heavy chain and determine the immunoreactivity of mAb. The antibody was purified with protein G Sepharose affinity chromatography filler. The purified antibody was detected by SDS-PAGE, and the effect of the antibody on LCMV was further examined by Western-blot. The results showed that the specific hybridoma cell lines secreting LCMV stably were successfully obtained, and the purified mAb.IFA,dot-blot showed that the obtained mAb could recognize the LCMV antigen. The immunoreactivity of the mAb heavy chain type to IgG2b, culture medium was 1.35 脳 103, which was identified by ELISA method. The purified antibody was confirmed by SDS-PAGE Coomassie brilliant Blue R250 after purified by protein G affinity chromatography. Western-blot indicated that the site of the effect of m Ab on LCMV was the surface glycoprotein antigen GP2. of virus particles. Conclusion the type of m Ab, antibody against LCMV virus surface glycoprotein antigen GP2 with high affinity is IgG2b..
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392-33

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