【作者】 胡青海；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2005， 博士

【摘要】 H5亚型高致病性禽流感(HPAI)是由H5亚型禽流感病毒(AIV)中的一些毒株引起的禽类高度致死性传染病,并威胁着人类的健康,被国际兽医局(OIE)列为A类疾病,在我国被列为一类动物传染病。国际上对HPAI的控制主要采取扑杀及其他生物安全措施,疫苗免疫接种也是控制疫情的关键措施之一。目前应用的油乳剂全病毒灭活疫苗虽然具有效果确实、保护率高、免疫期长和研制周期短等优点,但也存在许多缺陷,主要是灭活苗的使用影响疫情监测及不能产生粘膜免疫应答。DNA免疫能诱导机体产生特异性的细胞免疫、体液免疫和粘膜免疫,且不受母源抗体的干扰,被称为第三代疫苗。表达HA基因的AIV DNA疫苗只产生针对HA蛋白的抗体,不影响疫情监测,且受母源抗体的干扰小。但现有的H5亚型AIV DNA疫苗的免疫保护水平与油乳剂全病毒灭活疫苗相比仍有差距,需进一步优化,以提高免疫效力。许多研究表明,选择合适的DNA疫苗佐剂(如CpGDNA、细胞因子、趋化因子、HSP和C3d等)可在被接种动物体内很好地发挥免疫调节作用,不仅能增强免疫反应的强度,还可改变诱发的免疫反应类型,提高免疫力。本研究将构建鸡IL-2、IL-18或IFN-γ与H5亚型AIV HA基因共表达的DNA疫苗以及在真核表达质粒中嵌合CpG基序的DNA疫苗,然后以减毒鼠伤寒沙门氏菌SL7207运送这些真核表达质粒,以空白组、空载体组和禽流感油乳剂灭活疫苗免疫组作为对照,来评价鸡IL-2、IL-18、IFN-γ cDNA和CpG DNA在减毒沙门氏菌运送H5亚型禽流感核酸疫苗中的佐剂作用。 一些H5亚型禽流感DNA疫苗免疫后能提供部分或完全的免疫保护率,但未检测到血清抗体水平的升高。细胞免疫在这种疫苗免疫保护中究竟起多大作用?为了研究CD4~+或CD8~+T细胞在其中的作用以及CD3~+T细胞的绝对数量(或百分比)变化等,研制抗鸡CD3、CD4和CD8分子的单克隆抗体很有必要。更多还原

【Abstract】 H5 subtype of highly pathogenic avian influenza (HPAI), is a disease of poultry with high morbidity and mortality caused by specific influenza virus type A, and also threats to human health. HPAI is classified as one of the list A animal disease by OIE and China Ministry of Agriculture. Vaccination is frequently used to prevent and control the disease in some countries when the disease spreads widely. Current inactivated whole viral vaccines against HAPI in poultry have been proved to provide adequate protection against the disease. However, there are several disadvantages for these vaccines. Most of all, the antibodies induced by the vaccination interfere with the serological surveillance. A number of studies have shown that DNA vaccines can effectively induce cellular, humoral and mucosal immune responses against their encoded antigens. An HA DNA vaccine against HPAI only induces specific antibodies to HA, so the vaccine will not interfere with the serological surveillance. It can also overcome the interference of maternal antibodies. Although significant progress has been achieved in delivering HA DNA vaccines, immunogenicity of these DNA vaccines remains relatively low in chickens compared to inactivated whole viral vaccines. Several approaches have been used to influence not only the magnitude but also the type of immune response induced by DNA vaccines. Among these, the use of CpG DNA, cytokines, chemokines, costimulatory molecules, HSP, and c3d genes as adjuvants to DNA vaccines have been found to be important determinants of immunogenicity. One of main purposes of this paper was to evaluate the protective efficacy of HA DNA vaccines delivered by attenuated Salmonella typhimurium strain SL7207 strain with the co-expression of chicken IL-2, IL-18, IFN- γ, or built-in CpG DNA as adjuvant.In many studies on DNA vaccines against HPAI where no antibody was found, the vaccinated chickens were still partially to fully protected from challenge, indicating that an adaptive immune response other than humoral (presumably cellular immunity) was induced by the vaccination. There are no studies to date directly addressing the role of CD4+ or CD8+ T lymphocytes in the protection. To understand which subtypes of T cells were involved, It is necessary to develop mouse anti-chicken CD4 and CD8 mAbs.Chicken CD3, CD4 and CD8 are transmembrane glycoproteins on T lymphocytes, and they are also important cell surface markers of these cells. During antigen recognition, CD3 complex are involved in signal transduction, which ultimately leads to T-lymphocyte activation after the initial recognition steps. CD4 and CD8 molecules act as co-receptors for TCR in recognition of antigens. T cells that express CD4 recognize antigens in context with MHC class II, whereas T cells that express CD 8 recognize antigens in the context with MHC class I. Whether differences of the CD molecules at extracellular domain in chicken has any role in extending the antigen recognition capacity of TCR or in evading viral infections remain to be seen. There are rich resources of local breeds in China, and they are useful materials to study the polymorphism of chicken CD3, CD4 and CD8. In this study, chicken CD3, CD4 and CD8 a cDNA of Leghorn, ISA, and Chinese local breeds would be cloned and sequenced, and the polymorphism of these sequences would also be compared. These materials are useful to study the structures and functions of these molecules and to guild the applications of monoclonal antibodies against chicken CD3, CD4 or CD8 molecules.Anti-chicken CD3, CD4 or CD8 mAbs are useful tools to study the functions of T lymphocytes expressing CD3, CD4 or CD8 molecules and cellular immunity in chicken. It is difficult to obtain these mAbs using whole lymphocytes as immunogen. In this study, we attempt to prepare monoclonal antibodies against chicken CD4 and CD8 molecules based on DNA immunization strategy.1. Construction of expression vectors which co-expressing AIV HA with chicken IL-2, IL-18 or IFN- γ, or incorporating CpG DNA and HA into pVAXlChicken IL-2, IL-更多还原