【作者】 孔斌；

【导师】 胡敬东；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 白细胞介素18(interleukin-18, IL-18),又称为γ-干扰素(interferon-γ, IFN-γ)诱生因子(interferon-gamma-inducing factor, IGIF),是一种具有多向和多层次免疫调节功能的细胞因子,与肿瘤、变态反应性疾病、自身免疫性疾病、移植物抗宿主病等的发生发展密切相关,因此它在许多疾病的基础性研究和临床应用中有重要价值。目前,免疫抑制病给我国集约化养鸡业带来了很大危害,禽网状内皮组织增生症病毒(reticuloendotheliosis virus, REV)感染后可导致体液和细胞介导免疫应答的严重抑制已有很多报道,了解免疫抑制机理并制定提高鸡的免疫应答策略正成为当前的研究热点。本研究以禽网状内皮组织增生症病毒造成的免疫抑制为模型,对鸡白细胞介素18在免疫抑制中可能发挥的作用进行了初步探讨。研究内容主要分为两部分:1.成功建立了一种能够检测鸡白细胞介素18的SYBR GreenΙ荧光定量RT-PCR方法。根据GenBank上鸡白细胞介素18基因的序列,在保守区设计并合成各自特异引物,并以核糖体蛋白亚基4基因为内参,采用SYBR GreenⅠ染料以建立实时荧光定量RT-PCR检测方法。将白细胞介素18与核糖体蛋白亚基4基因克隆至pMD18-T载体上,以各阳性质粒作为标准品,构建标准曲线,并进行了熔解曲线分析。结果表明,该方法对鸡白细胞介素18 mRNA的扩增效率为107.16%,线性范围为10-2~10-6,相关系数为0.998,最低能检出100拷贝/μL;熔解曲线分析荧光定量RT-PCR产物在(80.4±0.6)℃出现单特异峰;从RNA提取到荧光定量PCR结束的检测周期只需4 h。本研究所建立的鸡白细胞介素18基因实时荧光定量RT-PCR方法灵敏度高、特异性强、检测周期短,为在转录水平对鸡IL-18的定量分析奠定了基础。2.实时荧光定量RT-PCR检测禽网状内皮组织增生症病毒感染肉鸡免疫器官白细胞介素18基因表达量的研究。为了研究鸡白细胞介素18基因在禽网状内皮组织增生症病毒感染肉鸡体内的表达水平,120只1日龄肉鸡随机分为2组,禽网状内皮组织增生症病毒处理组和生理盐水对照组。每个处理组分别在感染后7、14、21、28、35和42 d随机取6只鸡处死,迅速取脾脏、胸腺和法氏囊样品用于RNA提取。采用SYBR GreenⅠ染料实时荧光定量RT-PCR,比较处理组和对照组IL-18基因mRNA相对表达量的变化。结果显示,与对照组相比,处理组脾脏、胸腺和法氏囊中IL-18的分泌水平在感染后7d和14d均显著(P<0.05)升高,在21d、28d、35d和42d表达差异(P<0.05)也均显著。本实验为探讨禽网状内皮组织增生症病毒感染的免疫抑制机理打下了一定基础。更多还原

【Abstract】 IL-18(Interleukin-18, IL-18), also known as IFN-γinducible factor (interferon-gamma-inducing factor, IGIF), is a kind of multi-directional and multi-level immunomodulatory cytokine which is closely associated with the occurrence and development of carcinoma, allergy, auto-immune diseases, and graft-versus-host disease. Therefore, IL-18 will play an important role in basic research and clinical applications of many diseases. Now intensive immunosuppressive disease has brought great harm to the poultry industry in China. There have been many reports that REV(Reticuloendotheliosis virus, REV) infection can cause serious immunosupression of humoral and cell-mediated immune responses. Understanding the mechanism of immunosuppression and developing strategies to improve the immune responses of chickens have become the current research hotspots. In this study, immunosuppression caused by REV was used as a model and the role of chicken IL-18 in the immune suppression was discussed. And this study was divided into two parts:1. A real-time reverse-transcription PCR(RRT-PCR) based on SYBR Green I for detecting chicken IL-18 was established successfully. According to the chicken IL-18 gene sequence available in GenBank, a pair of primers was designed for developing a SYBR GreenⅠquantitative real-time PCR method to detect chicken IL-18 gene while the chicken ribosomal protein subunit 4(RPL4)gene was used as an internal control. To establish the standard curve, the positive plasmid of each gene served as a standard. The melting curve was also analyzed. The results showed that the amplification efficiency of this assay was 107.16%, and the linear range was 10-2~10-6 with good correlation coefficient of 0.998, as well as the detection limit reached 100 copies/μL. The melting curve presented a single peak at(80.4±0.6)℃. It only took 4 hours from extracting RNA to the end of real-time fluorescent quantitative RT-PCR. The developed real-time PCR assay could quickly detect IL-18 gene in expansion range with high efficiency, thus providing the basis for quantitative analysis of IL-18 gene expression.2. Study on IL-18 gene expression in the primary immune organs of broilers infected with REV by real-time quantitative RT-PCR. In order to study expression levels of IL-18 gene in broilers infected with reticuloendotheliosis virus(REV), 120 one-day-old broilers were randomly divided into 2 groups. The treatment group chickens was infected with REV while the control group was inoculated with saline. Six chickens of each group were randomly chosen and killed at day 7,14,21,28,35 and 42 post infection. Spleen, thymus and bursa were sampled for RNA extraction. SYBR GreenⅠq uantitative real-time PCR method was applied to compare the relative expression quantity of broiler IL-18 gene. The following results were obtained: IL-18 gene expression levels were significantly higher at day 7,14(P<0.05) and were significant at day 21,28,35 and 42(P<0.05) in the spleen, thymus and bursa in broiler chickens from treatment group than those in the control group. Our study laid a certain foundation for investigating the mechanism of immune suppression of REV infection.更多还原