本文選題：豬繁殖與呼吸綜合征病毒 + 抗體依賴性增強作用��； 參考：《中國農(nóng)業(yè)科學院》2015年碩士論文

【摘要】：豬繁殖與呼吸綜合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)引起的一種急性傳染病,是目前嚴重危害世界養(yǎng)豬業(yè)的一種重要病毒病,給養(yǎng)豬業(yè)造成了巨大經(jīng)濟損失。為了有效的控制該病的發(fā)生,眾多科學家從事疫苗的開發(fā)和研究。然而,由于動物豬體在感染PRRSV后所產(chǎn)生的免疫反應和疾病的發(fā)展過程受多種因素的影響,致使疫苗的免疫效果不盡人意;其中,病毒感染的抗體依賴性增強作用(antibody-dependent enhancement,ADE)是關(guān)鍵影響因素之一。ADE即指機體的抗體水平在未達到中和抗體濃度時,可協(xié)助病毒進入靶細胞,提高感染效率。目前,ADE作用已在多個科屬的40多種病毒的感染中發(fā)現(xiàn),如登革熱病毒、人免疫缺陷病毒、西尼羅河病毒等。與PRRSV相似的是這些病毒多表現(xiàn)為嗜好在免疫細胞中繁殖,容易引發(fā)宿主的持續(xù)性感染。常規(guī)疫苗免疫防制這類具有ADE作用的病毒病時常常難以奏效,動物免疫后,對該病毒的易感性反而會增加。因此,有必要利用病毒模型,探討ADE在這類病毒中的致病機制。因此,為了更有效的防控PRRS的發(fā)生,本課題即從ADE角度出發(fā),闡釋了PRRSV的致病機理。本研究發(fā)現(xiàn)當亞中和活性的PRRSV特異性抗體存在時,PRRSV體外感染豬肺泡巨噬細胞(porcine alveolar macrophages,PAMs)能力增強,即,亞中和活性的抗體可促進病毒的感染和釋放。已有的報道表明,介導ADE效應的細胞表面分子有三種Fcγ受體(FcγRs),即:FcγRⅠ(CD64)、FcγRⅡ(CD32)和FcγRⅢ(CD16)。通過運用熒光定量PCR方法,本研究發(fā)現(xiàn)CD16在PAMs的轉(zhuǎn)錄水平遠遠高于CD32和CD64;同時,流式細胞術(shù)和Western blotting的結(jié)果也表明CD16在PAMs上高效表達。因此,本研究對豬源CD16在PRRSV的ADE效應中發(fā)揮的作用進行研究。首先,運用抗CD16的單克隆抗體特異性阻斷PAMs表面的CD16,探究CD16在PRRSV的ADE效應中是否發(fā)揮作用。結(jié)果發(fā)現(xiàn),CD16功能被抗體特異性阻斷后,PRRSV亞中和活性的抗體引發(fā)的PRRSV的ADE效應顯著降低,說明PAMs表面的CD16能夠介導PRRSV的ADE。由于PAMs表面存在三種不同的FcγRs,為了特異性研究CD16,本研究在無FcγRs表達的HEK293-T細胞和COS-7細胞上特異性表達豬源CD16,進一步驗證CD16介導的ADE效應。研究結(jié)果發(fā)現(xiàn),PRRSV-抗體免疫復合物可特異性吸附在HEK293-T/CD16細胞和COS-7/CD16細胞,進而使得PRRSV內(nèi)化進入細胞。最后,本研究還發(fā)現(xiàn)內(nèi)化的PRRSV可以在這些細胞內(nèi)發(fā)生復制,并釋放出具有活性的子代病毒。此外,本研究還證明豬源CD16的高效表達需要和Fc受體γ鏈的協(xié)同表達。本研究對PRRSV的ADE效應提出新的機制,即首次證明CD16能介導PRRSV的ADE,為進一步揭示PRRSV的致病機制提供新理論。本研究提示當動物機體存在亞中和活性的抗體時,PRRSV可能對CD16陽性的細胞具有一定的趨化性,使得PRRSV在感染的過程中有更廣泛的分布性,本研究為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.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.651

【參考文獻】

相關(guān)期刊論文 前1條

1 謝英,單天錫;豬繁殖與呼吸綜合征研究進展[J];中國獸醫(yī)雜志;2001年01期

，

本文編號：2104378