犬细胞免疫因子RT-qPCR检测方法的建立及应用

发布时间：2018-01-31 12:53

本文关键词： 荧光定量PCR IL-4 IL-10 IL-13 IFN-α 免疫评价犬三联活疫苗　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：实时荧光定量PCR技术是在PCR反应体系中加入荧光探针或者荧光染料,利用荧光信号的积累,实时监测整个PCR的反映进程,通过制作出的标准曲线,对未知模版进行定量分析。因其具有操作简便、快速高效、高敏感性等特点,被广泛应用于分子诊断、分子生物研究、动物及食品安全检测等方面。本实验分离了犬全血中的外周血淋巴细胞,通过PCR成功扩增出IL-4、IL-10、IL-13、IFN-α等几种细胞免疫因子的基因片段,连接pMD-18T载体,转化到DH5α感受态细胞中,在Amp抗性培养基上进行筛选、PCR和测序等验证,构建了标准品质粒。最后成功建立了检测犬全血中IL-4、IL-10、IL-13、IFN-α等细胞因子的实时荧光定量PCR的方法,该方法的模版在102~108拷贝/μL范围内呈现良好的线性关系,相关系数R2均达到0.992以上。利用该方法对犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬腺病毒Ⅰ型(CAV-1)三联活疫苗免疫比格犬后的细胞免疫水平进行了评价。实验结果显示:犬三联活疫苗免疫犬后,免疫犬外周血淋巴细胞与空白对照外周血淋巴细胞相比,IL-4、IL-10、IL-13、IFN-α等几种细胞免疫因子mRNA水平在3~14天之间均显著升高(p0.05);空白对照组的4种细胞免疫因子mRNA水平没有明显差异(p0.05)。以上实验结果表明,本研究建立的实时荧光定量PCR方法确实可行,IL-4、IL-10、IL-13、IFN-α等细胞免疫因子可以作为细胞免疫评价指标之一。
[Abstract]:Real-time fluorescence quantitative PCR technology is to add fluorescent probes or fluorescent dyes into the PCR reaction system, using the accumulation of fluorescence signals, real-time monitoring the whole PCR reaction process, through the preparation of the standard curve. Quantitative analysis of unknown templates has been widely used in molecular diagnosis and molecular biology research because of its advantages of simple operation, high efficiency, high sensitivity and so on. The peripheral blood lymphocytes were isolated from the whole blood of dogs, and IL-4 IL-10 IL-13 was amplified successfully by PCR. The gene fragments of IFN- 伪 and other cytokines were ligated into pMD-18T vector and transformed into DH5 伪 competent cells, and screened on Amp resistant medium. The standard plasmid was constructed by PCR and sequencing. Finally, the detection of IL-4, IL-10 and IL-13 in the whole blood of dogs was successfully established. IFN- 伪 and other cytokines of real-time fluorescence quantitative PCR method, the template of this method in 102108copies / 渭 L showed a good linear relationship. The correlation coefficient R2 was above 0.992. The method was used to treat canine distemper virus (CDV) and canine parvovirus (CPV). The cellular immunity level of Beagle dogs immunized with CAV-1 triple live vaccine was evaluated. The results showed that the immunized dogs were immunized with three live vaccines. The peripheral blood lymphocytes of immunized dogs were compared with the peripheral blood lymphocytes of blank control. The level of IFN- 伪 and other cytokines mRNA increased significantly between 3d and 14d. There was no significant difference in the levels of mRNA among the four cytokines in the blank control group. The above results showed that the real-time fluorescent quantitative PCR method established in this study is feasible for IL-4. Cytokines such as IL-10, IL-13, IFN- 伪 can be used as one of the indexes for evaluating cellular immunity.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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