猪FcγRⅢ介导PRRSV抗体依赖增强作用研究

Porcine FcγRⅢ and Its Role in Antibody-dependent Enhencement of PRRSV Infection

作者: 专业:动物学 导师:王爱萍 年度:2010  院校: 郑州大学

Keywords

Fcγreceptors, co-transfection, PRRSV, ADE
        IgG Fc受体(Fcγreceptors,FcγRs)可特异亲和免疫球蛋白IgG(IgG, Immunoglobulin G)Fc片段,是非常重要的免疫细胞表面分子,在机体免疫防御中有很重要的作用,是细胞免疫和体液免疫的桥梁。研究发现FcγRs具有修复炎性损伤、吞噬作用、抗体依赖细胞毒作用、释放炎性介质和调节B细胞活性等功能,是哺乳动物免疫应答的重要组成部分。FcγRs是治疗自身免疫性疾病、过敏、癌症等免疫疾病理想的药物靶标。家畜FcγRs的研究比人与小鼠滞后,但近年来也有很大进展,一些家畜FcγRs基因先后被克隆,生物学特性方面研究也取得进步。猪IgG Fc受体由三类分子组成:FcγRⅠ(CD64).FcγRⅡ(CD32).FcγRⅢ(CD 16).其中FcγRⅢ(CD16)为50-80 kDa的膜糖蛋白,在巨噬细胞、肥大细胞和PMN细胞中都有表达,是唯一在NK细胞上表达的Fc受体。FcγRⅢ有两个Ig样胞外区结构域,是中低亲和力受体。猪FcγRⅢA是首个克隆的家畜FcγRⅢ基因,在多形核细胞(PMN, polymorphonuclear)中为45 kDa糖蛋白,其胞内区与FcRγ链或其它亚基相连,只有通过共转染FcRγ链,才能在细胞表面表达FcγRⅢA.猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的以母猪流产和仔猪呼吸障碍为主要特征的传染病,是困扰中国和世界养猪业的主要传染病。PRRSV具有抗体依赖性增强作用(ADE)、病毒容易变异、存在持续感染性及免疫抑制等免疫特性,其中ADE是困扰PRRS防控的最主要问题。目前有关PRRS ADE的分子机制研究较少。ADE主要由Fc受体介导,为研究猪FcγRⅢ在介导PRRS ADE中的作用,本研究构建了猪FcγRⅢ和FcRγ链基因真核表达质粒,共转染Marc-145细胞,通过潮霉素B和G418连续双抗性筛选和克隆获得一株稳定表达猪FcγRⅢ的Marc-145细胞系。用PRRSV BJ-4株与1600倍稀释的抗PRRSV IgG等体积混合后感染克隆化的Marc-145细胞系,与空白对照相比,有抗体存在时PRRSV感染获得的PRRSV病毒量更高,证明了猪FcγRⅢ能够介导PRRSV抗体依赖增强作用.本试验通过研究PRRSV和Fc受体之间的相互作用,初步揭示PRRSV抗体依赖增强作用的分子机制,为研制开发新型疫苗、新型免疫佐剂和抗病毒药物提供理论依据,为进一步研究打下基础。
    IgG Fc receptors (FcyRs), which are widely distributed on the surface of well-known immune cells, are an important family of receptor molecules. They can bind with the immunoglobulin G (IgG) fragments specifically, providing a link between cell-mediated immune and humoral immune response, and play a key role in the body’s immune defense system. The latest study revealed that FcyRs are also ideal drug targets in treating allergic and autoimmune diseases, in that their important immune function in regulating positive and negative cell response.In recent few years, research on livestock FcyRs has made great progress. The FcyRs genes in main livestock species have been cloned and the biologically functions were characterized. Porcine FcyRs are consisted of three types of molecules, such as FcyRI (CD64), FcyRII (CD32) and FcyRIII(CD16). FcyRIII is a kind of 50-80 kD glycoprotein expressed on the membrane of alveolar macrophges cells, mast cells and polymorphnuclear (PMN) cells. The porcine FcyRIIIA complex are 45 kDa glycoprotein on the PMN cells, and their intracellular domains always connect with FcR y chain or other subunits. So the in vitro expression of FcyRIIIA complex on membrane must depend on the co-transfection of FcyRIIIA and FcR y chain genes.Porcine reproductive and respiratory syndrome (PRRS) is an infectious viral disease caused by PRRSV and characterized by the abortion of pregnant sow and breathing obstacle of pig. Recent years, the infection and spread of virulent strain of PRRSV has caused great damage to the farming of swine all over the world. Antibody dependent enhancement (ADE) is a key impediment for developing efficient vaccine.To investigate the molecular mechanism of ADE, two eukaryotic expression plasmid containing FcyRIII and FcRy chain genes were individually constructed and co-transfected into Marc-45 cells. The Mar-45 cells which can stably expressed FcyRⅢcomplex were selected and cloned by the bi-antigenic against hygromycin B and G418. When the cloned Marc-145 cells was infeted with BJ-4, the well-known standard strain of PRSSV and the 1:1600-diluted anti-PRRSV IgG mixture, more virus was harvested than the control group without IgG. This indicated that FcyRIII complex could mediate ADE in the infection of PRSSV. In the study, the preliminary molecular mechanism of ADE was investigated by studying the interaction of PRRSV and FcyRs. The result not only provided theoretical basis for new vaccine, immune adjuvant and anti-virus drugs, but also help to search for other pathogenic mechanisms of important diseases.
        

猪FcγRⅢ介导PRRSV抗体依赖增强作用研究

中文摘要4-5
Abstract5-6
目录7-11
第一章 前言11-19
    1.1 Fc受体的分类11
    1.2 FcγRs亚类的生物学特性及家畜FcγRs研究进展11-13
        1.2.1 FcγRⅠ(CD64)11-12
        1.2.2 FcγRⅡ(CD32)12-13
        1.2.3 FcγRⅢ(CD16)13
        1.2.4 moFcγRⅣ13
        1.2.5 boFcγ2R13
    1.3 FcγR的免疫学功能研究进展13-15
        1.3.1 调节抗体介导的免疫细胞活化14
        1.3.2 介导抗体亚类的不同免疫功能14
        1.3.3 介导细胞吞噬作用14-15
        1.3.4 介导ADCC作用15
        1.3.5 介导树突状细胞抗原提呈15
    1.4 PRRSV的研究进展15-17
        1.4.1 PRRSV15-16
        1.4.2 PRRSV基因组结构16
        1.4.3 PRRSV免疫学研究进展16-17
    1.5 ADE作用的研究进展17-19
第二章 转染Marc-145细胞条件优化19-37
    2.1 材料19-23
        2.1.1 质粒、菌种19
        2.1.2 细胞19
        2.1.3 酶、主要试剂19-20
        2.1.4 试剂配制20-22
        2.1.5 主要仪器22-23
    2.2 方法23-31
        2.2.1 GFP基因片段的扩增23
        2.2.2 GFP重组真核表达质粒的构建23-27
        2.2.3 GFP转染MARC-145细胞27-29
        2.2.4 转染条件优化29-31
    2.3 结果31-36
        2.3.1 GFP片段的扩增31
        2.3.2 GFP真核表达质粒的构建31-33
        2.3.3 稳定转染Marc-145细胞33
        2.3.4 质粒转染Marc-145细胞条件优化33-36
    2.4 讨论36-37
第三章 稳定表达猪FcγRⅢ共转染细胞株的建立37-57
    3.1 材料37
        3.1.1 细胞37
        3.1.2 酶、主要试剂37
        3.1.3 试剂配制37
    3.2 方法37-47
        3.2.1 猪肺泡巨噬细胞(PAM)的采集37-38
        3.2.2 PAM细胞cDNA的获取38-39
        3.2.3 猪FcγRⅢ基因片段的克隆39-40
        3.2.4 真核表达质粒pcDNA3.1-FcγRⅢ的构建40-41
        3.2.5 猪γ链基因片段的克隆41-42
        3.2.6 真核表达质粒PIREShyg3-γ的构建42-43
        3.2.7 稳定转染细胞系的建立43-46
        3.2.8 共转染细胞系的RT-PCR鉴定46
        3.2.9 共转染细胞系的玫瑰花环试验46
        3.2.10 共转染细胞系的流式鉴定46-47
    3.3 结果与分析47-55
        3.3.1 PAM总RNA的提取47-48
        3.3.2 猪FcγRⅢ基因片段的扩增48
        3.3.3 猪γ基因片段的扩增48-49
        3.3.4 真核表达质粒pcDNA3.1-FcγRⅢ的构建49-50
        3.3.5 真核表达质粒PIREShyg3-γ的构建50
        3.3.6 共转染稳定表达细胞系的鉴定50-55
    3.4 讨论55-57
第四章 poFcγRⅢ介导PRRSV抗体依赖增强作用(ADE)57-63
    4.1 材料57
        4.1.1 病毒和细胞57
        4.1.2 主要试剂57
    4.2 方法57-59
        4.2.1 病毒的增殖57
        4.2.2 病毒TCID_(50)的测定57-58
        4.2.3 猪FcγRⅢ介导PRRSV抗体依赖增强作用58
        4.2.4 兔抗猪FcγRⅢ多抗效价的测定58-59
        4.2.5 rsFcγRⅢ抗体对转染细胞系介导PRRSV ADE的阻断59
    4.3 结果59-62
        4.3.1 poFcγRⅢ介异PRRSV ADE作用59-61
        4.3.2 rspoRⅢ多抗效价的测定61
        4.3.3 抗rsFcγRⅢ对PRRSV ADE的阻断作用61-62
    4.4 讨论62-63
第五章 全文总结63-64
参考文献64-69
作者简历69
硕士在读期间撰写和已发表的论文69-70
附录A:英文缩略词表70-71
附录B:质粒图谱71-73
致谢73
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