猪CD16及其介导PRRSV抗体依赖性增强作用研究

发布时间：2018-07-07 08:27

本文选题：猪繁殖与呼吸综合征病毒 + 抗体依赖性增强作用　；参考：《中国农业科学院》2015年硕士论文

【摘要】：猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)引起的一种急性传染病,是目前严重危害世界养猪业的一种重要病毒病,给养猪业造成了巨大经济损失。为了有效的控制该病的发生,众多科学家从事疫苗的开发和研究。然而,由于动物猪体在感染PRRSV后所产生的免疫反应和疾病的发展过程受多种因素的影响,致使疫苗的免疫效果不尽人意;其中,病毒感染的抗体依赖性增强作用(antibody-dependent enhancement,ADE)是关键影响因素之一。ADE即指机体的抗体水平在未达到中和抗体浓度时,可协助病毒进入靶细胞,提高感染效率。目前,ADE作用已在多个科属的40多种病毒的感染中发现,如登革热病毒、人免疫缺陷病毒、西尼罗河病毒等。与PRRSV相似的是这些病毒多表现为嗜好在免疫细胞中繁殖,容易引发宿主的持续性感染。常规疫苗免疫防制这类具有ADE作用的病毒病时常常难以奏效,动物免疫后,对该病毒的易感性反而会增加。因此,有必要利用病毒模型,探讨ADE在这类病毒中的致病机制。因此,为了更有效的防控PRRS的发生,本课题即从ADE角度出发,阐释了PRRSV的致病机理。本研究发现当亚中和活性的PRRSV特异性抗体存在时,PRRSV体外感染猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs)能力增强,即,亚中和活性的抗体可促进病毒的感染和释放。已有的报道表明,介导ADE效应的细胞表面分子有三种Fcγ受体(FcγRs),即:FcγRⅠ(CD64)、FcγRⅡ(CD32)和FcγRⅢ(CD16)。通过运用荧光定量PCR方法,本研究发现CD16在PAMs的转录水平远远高于CD32和CD64;同时,流式细胞术和Western blotting的结果也表明CD16在PAMs上高效表达。因此,本研究对猪源CD16在PRRSV的ADE效应中发挥的作用进行研究。首先,运用抗CD16的单克隆抗体特异性阻断PAMs表面的CD16,探究CD16在PRRSV的ADE效应中是否发挥作用。结果发现,CD16功能被抗体特异性阻断后,PRRSV亚中和活性的抗体引发的PRRSV的ADE效应显著降低,说明PAMs表面的CD16能够介导PRRSV的ADE。由于PAMs表面存在三种不同的FcγRs,为了特异性研究CD16,本研究在无FcγRs表达的HEK293-T细胞和COS-7细胞上特异性表达猪源CD16,进一步验证CD16介导的ADE效应。研究结果发现,PRRSV-抗体免疫复合物可特异性吸附在HEK293-T/CD16细胞和COS-7/CD16细胞,进而使得PRRSV内化进入细胞。最后,本研究还发现内化的PRRSV可以在这些细胞内发生复制,并释放出具有活性的子代病毒。此外,本研究还证明猪源CD16的高效表达需要和Fc受体γ链的协同表达。本研究对PRRSV的ADE效应提出新的机制,即首次证明CD16能介导PRRSV的ADE,为进一步揭示PRRSV的致病机制提供新理论。本研究提示当动物机体存在亚中和活性的抗体时,PRRSV可能对CD16阳性的细胞具有一定的趋化性,使得PRRSV在感染的过程中有更广泛的分布性,本研究为PRRSV疫苗的研究提供了新思路。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus). It is an important viral disease that seriously endangers the world's pig industry. It has caused a huge amount of swine industry to the pig industry. In order to effectively control the occurrence of the disease, many scientists have been engaged in the development and research of vaccines. However, the immune response and the development of the disease caused by the infection of PRRSV in animal pigs are affected by many factors, and the immune effect of the vaccine is unsatisfactory, and the antibody dependent enhancement of the virus infection (a Ntibody-dependent enhancement, ADE) is one of the key influencing factors,.ADE, which means that the level of the body's antibody can help the virus enter the target cell and improve the infection efficiency when it does not reach the neutralization antibody concentration. At present, the ADE effect has been found in more than 40 viruses of multiple families, such as dengue virus, human immunodeficiency virus, West Nile. Similar to PRRSV, these viruses are similar to those of the virus that reproduce in immune cells and easily lead to persistent infection of the host. Conventional vaccines are often difficult to perform when they are immune to ADE, and the susceptibility to the virus increases after being immune to animals. Therefore, it is necessary to use the virus model to explore A. The pathogenesis of DE in this kind of virus. Therefore, in order to prevent and control the occurrence of PRRS more effectively, this topic is to explain the pathogenesis of PRRSV from the ADE point of view. This study found that when the PRRSV specific antibody of subneutralizing active PRRSV is present, PRRSV infection in porcine alveolar macrophages (porcine alveolar macrophages, PAMs) is enhanced in vitro, that is, Subneutralizing antibodies can promote the infection and release of the virus. It has been reported that there are three kinds of Fc gamma receptors (Fc gamma Rs) in the cell surface molecules mediated by the ADE effect, namely, Fc gamma R I (CD64), Fc gamma R II (CD32) and Fc gamma R. The results of flow cytometry and Western blotting also indicate that CD16 is highly expressed on PAMs. Therefore, this study studies the role of the pig source CD16 in the ADE effect of PRRSV. First, using the monoclonal antibody against CD16 to specifically block the CD16 of the PAMs surface, explore whether CD16 plays a role in the PRRSV effect. The results have been found, After the function of CD16 was specifically blocked by antibody, the ADE effect of PRRSV induced by PRRSV subneutralizing antibody was significantly reduced, indicating that CD16 on the PAMs surface could mediate PRRSV ADE. due to the existence of three different Fc gamma Rs on the PAMs surface. CD16, a pig source, further verified the ADE effect mediated by CD16. The results showed that the PRRSV- antibody immune complex could be specifically adsorbed in HEK293-T/CD16 cells and COS-7/CD16 cells, thus making PRRSV internalized into cells. Finally, the present study found that the internalized PRRSV could reproduce within these cells and release the active offspring. In addition, this study also demonstrated that the efficient expression of the pig source CD16 needs to be co expressed with the Fc receptor gamma chain. This study provides a new mechanism for the ADE effect of PRRSV, which is the first evidence that CD16 can mediate the ADE of PRRSV, which provides a new theory to further reveal the pathogenesis of PRRSV. In vivo, PRRSV may have certain chemotaxis to CD16 positive cells, which makes PRRSV more widely distributed in the process of infection. This study provides a new way of thinking for the research of PRRSV vaccine.
【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】：2015
【分类号】：S852.651

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