【作者】 郑玉姝；

【导师】 赵宏坤； 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2006， 博士

【摘要】 多种病毒感染都可能引起禽类的免疫抑制。如马立克氏病病毒(MDV)、禽网状内皮增生病病毒(REV)、禽J亚群白血病病毒(ALV-J)、鸡传染性贫血病病毒(CAV)、禽呼肠孤病毒(ARV)、新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)等。流行病学调查表明,免疫抑制性病毒感染在鸡群中相当普遍,而且这些病毒造成的二重感染及多重感染可能导致更为严重的免疫抑制。免疫抑制病给我国集约化养禽业带来了很大危害。但迄今为止,对上述病毒诱发免疫抑制的机理,特别是细胞因子在免疫抑制性病毒感染、发病及免疫反应中发挥的作用还不清楚。有研究表明,一些病原引起的免疫抑制可能与细胞因子的分泌水平和表达异常有关。其中对于免疫应答的重要调节因子——γ-干扰素(IFN-γ)通常采用反转录-聚合酶链式反应(RT-PCR)在mRNA水平上进行检测,但这种方法只能间接反映病毒感染对脾白细胞分泌IFN-γ的影响。本研究则是采用双抗体夹心酶联免疫吸附试验(ELISA)对REV、MDV、CAV和ARV感染SPF鸡的脾白细胞分泌的IFN-γ水平进行了直接定量研究。分别在REV、MDV、CAV和ARV单独感染和共感染鸡后的不同时间,采集脾脏制备脾白细胞,并进行培养,并比较在脾白细胞中IFN-γ的分泌水平,以此阐明IFN-γ在REV、MDV、CAV和ARV感染引起的免疫抑制过程中可能发挥的作用。采用生物素-链霉亲和素系统放大的双抗体夹心酶联免疫吸附方法,以鼠抗鸡IFN-γ单克隆抗体(mAb)为包被抗体,以针对不同抗原表位的生物素化mAb作为检测抗体对IFN-γ进行定量测定,并对ELISA试验影响因素进行了探讨。结果表明,在10-1000 pg/mL的浓度范围内,IFN-γ标准品浓度与吸光度值线性关系良好,相关系数为r=0.9921。以pH 7.2的磷酸盐缓冲液(PBS)为包被液包被12 h,以1%牛血清白蛋白(BSA)为封闭液,室温封闭2 h,并以含1% BSA的PBS-Tween(PBST)为样品稀释液,降低了非特异性吸附,提高了灵敏度。正常情况下,IFN基因表达处于抑制状态,在诱生剂的诱导作用下可以进行表达。因此,本研究采用双抗体夹心ELISA对培养的不同种类细胞在不同诱生剂作用下IFN-γ的分泌水平进行了定量检测,并对影响IFN-γ产量的条件进行了优化。结果表明:在刀豆蛋白A(ConA)、植物凝集素(PHA)和ARV这3种诱生剂中,ARV诱生能力最强,更多还原

【Abstract】 Many viruses are likely to cause immunosuppression in chickens, such as Marek’s disease virus (MDV), reticuloendotheliosis virus (REV) subgroup J avian leucosis virus (ALV-J), chicken anemia virus (CAV), reovirus (ARV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and etc. Epidemiological investigation indicated that immunosuppressive viral infections are very common in chickens. Moreover, immunosuppression could become more serious when dual or multiple viral infections and their interactions happened. Immunosuppressive diseases have brought our poultry industry great endangerment.However, the mechanism of virus-induced immunosuppression has been unknown to us, especially the role of cytokines in avian immunosuppressive viral infection, pathogenesis and immune response. Many reports demonstrated immunosuppression induced by some pathogen were related to the secreting levels and abnormal expression of cytokines. Most researchers measured interferon-gamma(IFN-γ) mRNA by reverse transcript-polymerase chain reaction(RT-PCR)and only showed the stimulation to IFN-γsecreting from splenic leukocyte by viral infection indirectly. In this study, double-antibody sandwich enzyme linked immunosorbent assay(ELISA) was established based on commercial chicken IFN-γmonoclone antibody(mAb) and IFN-γstandard, and directly quantitative measured the levels of IFN-γfrom splenic leukocyte of specific pathogen-free chickens infected by REV, MDV, CAV and ARV. Spleens were collected, Splenic leukocytes were prepared and cultured at various times after infection with REV, MDV, CAV and ARV and compare them with those in uninfected age-matched controls, so as to delineate the potential role of IFN-γin the mechanism of virus-induced immunosuppression.Chicken IFN-γwas measured by biotin-avidin-amplified double-antibody sandwich ELISA更多还原