肠道病毒71型的分离检测及灭活疫苗免疫原性研究

发布时间：2018-01-31 18:00

本文关键词： 肠道病毒分离检测活疫苗免疫原性研究　出处：《昆明理工大学》2012年硕士论文　论文类型：学位论文

【摘要】：手足口病(hand, foot and mouth disease, HFMD)是由肠道病毒引发的一种疾病,主要在5岁以下儿童中流行,传染性强,可引发多种中枢神经系统并发症,导致患儿死亡。目前,引发手足口病的病毒主要为柯萨奇病毒A组16型(CoxA16)(?)口肠道病毒71型(EV71)。由于手足口病发病迅速、在发病初期,临床表现不明显,难以在发病的初期进行及时的诊断,目前针对手足口病的治疗只限于发病后的对症治疗,没有有效的抗病毒药物,也没有用于预防该病的疫苗。随着近几年由EV71病毒引发的手足口病在我国的大规模爆发,我国很可能已经成为了该病潜在爆发区[3,4,5,61, EV71是引起手足口病的重要病原微生物,特别是重症感染、致死性感染的主要病因。在灭活疫苗、减毒活疫苗、亚单位疫苗、DNA疫苗、病毒样颗粒疫苗、转基因动物疫苗中,灭活疫苗因具备较为完备的抗原表位和较为简单的制备条件,最具开发潜力的疫苗形式,然而EV71的特点之一就是具有高度的变异性,分离得到最新流行的病毒是制备灭活疫苗的基础。本课题的第一部分主要包括处理粪便、分离病毒；RT-PCR分群鉴定毒种；EV71噬斑形成实验可行性预实验；显微挑斑法纯化病毒；RT-PCR鉴定是否分离成功；病变形态学筛选EV71；VP1基因的菌落PCR和酶切鉴定；VP1基因全序测定及进化树的构建；全序测定的菌落PCR鉴定、酶切鉴定；全序测定；EV71电镜观察；EV71免疫荧光实验。第二部分主要包括EV71MOI检测；无血清维持培养Vero NaHCO3含量值条件摸索；EV71在细胞和上清中的分布；无血清维持培养EV71可行性研究,Vero细胞有／无血清维持培养EV71上清中病毒一步动力曲线检测；Vero细胞无血清维持培养EV71上清和细胞中病毒增殖动力曲线；氯仿抽提、超声对EV71滴度的影响；EV71传代稳定性研究。第三部分主要包括细胞、病毒增殖曲线,pH变化情况；纯化、灭活病毒；EV71灭活疫苗免疫后总抗体的检测；EV71灭活疫苗中和抗体检测。本文创新性改进了病毒分离方法,明显提高了病毒分离纯化效率,通过这种方法成功的将EV71和CA16分离开来。经过分型鉴定证明分离到的EV71为C4a亚型,并成功获得全长序列和电镜照片。进一步检测了MOI、无血清培养pH条件、病毒在细胞内外的增殖曲线等生物学特性。最后,将EV71灭活后免疫动物,检测到了较高滴度的免疫原性和中和抗体效价。
[Abstract]:Hand, foot and mouth disease (HFMD) is a disease caused by enterovirus, mainly in children under 5 years of age. Infectious, can cause a variety of central nervous system complications, leading to child death. At present, the main cause of hand, foot and mouth disease is Coxsackie virus group A CoxA16? Due to the rapid onset of hand, foot and mouth disease, the clinical manifestation is not obvious at the beginning of the disease, so it is difficult to make timely diagnosis in the early stage of the disease. At present, the treatment of hand, foot and mouth disease is limited to the symptomatic treatment after onset, there is no effective antiviral drugs. There is no vaccine to prevent the disease. With the massive outbreak of HFMD caused by EV71 virus in China in recent years, China is likely to have become a potential outbreak of the disease. [3,4,5,61, EV71 is an important pathogen of hand, foot and mouth disease (HFMD), especially the main cause of severe infection and fatal infection, including inactivated vaccine, live attenuated vaccine and subunit vaccine. Virus-like granular vaccine, transgenic animal vaccine, inactivated vaccine because of relatively complete antigen epitopes and relatively simple preparation conditions, the most potential form of vaccine development. However, one of the characteristics of EV71 is its high variability, the isolation of the latest prevalent virus is the basis for the preparation of inactivated vaccines. The first part of this subject mainly includes the disposal of feces and isolation of viruses. RT-PCR group identification of virus species; EV71 plaque formation experiment feasibility pretest; The virus was purified by microblotting method. RT-PCR was used to identify whether the separation was successful or not. EV71 was screened by pathological morphology. Colony PCR and enzyme digestion of VP1 gene; Total sequence determination of VP1 gene and construction of phylogenetic tree; Total sequence determination of colony PCR identification, enzyme digestion identification; Total sequence determination; EV71 electron microscopy; EV71 immunofluorescence assay. The second part mainly includes the detection of EV71MOI; The conditions of Vero NaHCO3 content in serum-free culture were explored. The distribution of EV71 in cells and supernatants; Feasibility study of serum-free maintenance culture of EV71 virus in supernatant of EV71 cultured with or without serum-free was detected by one step dynamic curve. Vero cells maintained the EV71 supernatant and cell proliferation dynamics curve without serum. The effect of ultrasound on the titer of EV71 by chloroform extraction; Study on the stability of EV71 passage. The third part mainly includes cell, virus proliferation curve and pH change; Purification, inactivation of virus; Detection of total antibody after immunization with inactivated EV71 vaccine; Detection of neutralizing antibody against EV71 inactivated vaccine. In this paper, the virus isolation method was innovatively improved, and the efficiency of virus isolation and purification was improved obviously. EV71 and CA16 were successfully separated by this method. The isolated EV71 was identified as C4a subtype. The full length sequence and electron microscope photos were obtained successfully. The biological characteristics of moi, the pH condition of serum-free culture, the proliferation curve of the virus in and out of cells were further detected. Finally, the EV71 was inactivated to immunize the animals. High titer immunogenicity and neutralizing antibody titer were detected.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1;R725.1

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