应用蛋白芯片和免疫PCR技术高效检测汉滩病毒的研究

发布时间：2018-03-21 11:04

本文选题：汉坦病毒　切入点：核蛋白　出处：《中国科学院研究生院（武汉病毒研究所）》2005年硕士论文　论文类型：学位论文

【摘要】：肾综合征出血热(HFRS)是我国广泛流行的一种人兽共患的急性病毒性传染病,目前该病在我国的发病率占全球总数的90%,病死率为3%～10%,是除病毒性肝炎外危害最大的一类病毒性疾病。肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属病毒引起的自然疫源性传染病,临床表现复杂多样,病情变化较快,给临床诊断带来一定困难。目前,血清学检测方法仍然是HFRS的主要检测手段,但是以往的血清学诊断方法,如间接免疫荧光试验、斑点免疫结合试验、带状免疫斑点试验等敏感性和特异性不够高,因此有必要建立一种高灵敏的、高特异的、简便的HFRS辅助诊断技术。汉坦病毒核蛋白抗原(NP)在人体液免疫反应中起重要作用,据此本研究针对上述需求发展了由单链抗体(scFv)介导的免疫荧光蛋白质芯片和免疫PCR检测方法。将抗汉滩病毒核蛋白的单链抗体基因与碱性磷酸酶基因融合,获得了碱性磷酸酶介导的检测抗体;将单链抗体用Cy3 标记以及将抗体基因C 末端融合SBP基因,表达出的融合蛋白可以与链霉亲和素修饰的荧光纳米颗粒结合,实现了在蛋白质芯片上检测汉滩病毒核蛋白。本研究由单链抗体介导的免疫荧光蛋白质芯片检测方法,可在短时间内检测到10 pg/ml 的核蛋白;通过应用免疫-PCR方法检测到了更低浓度的核蛋白(10 fg/ml),这些方法的灵敏度都高于传统的ELISA(1 μg/ml)检测方法。另外,我们初步尝试了应用荧光高聚物实施NP 的均相检测,相比于其他方法,它是非常简便迅速,几分钟内可检测到10 ng/ml的核蛋白。
[Abstract]:Hemorrhagic fever with renal syndrome (HFRSs) is an acute viral infectious disease with zoonotic disease, which is widely prevalent in China. At present, the incidence of the disease in China accounts for 90% of the global total, and the mortality rate is 3100.It is the most harmful virus disease besides viral hepatitis. Hemorrhagic fever with renal syndrome (HFRSs) is caused by Bunyaviridae Hantavirus. Natural infectious diseases, At present, serological detection is still the main method for the detection of HFRS, but the previous serological diagnosis methods, such as indirect immunofluorescence test, are difficult to diagnose. The sensitivity and specificity of dot immunobinding assay and band immunodot test are not high enough, so it is necessary to establish a highly sensitive, highly specific, and so on. The hantavirus nucleoprotein antigen (HFRS) plays an important role in human humoral immune response. In view of the above requirements, a novel immunofluorescent protein chip mediated by scFvv-mediated immunofluorescence protein chip and immunological PCR detection method were developed. The detection antibody mediated by alkaline phosphatase was obtained by fusion of the single chain antibody gene against Hantaan virus nucleoprotein with alkaline phosphatase gene, and SBP gene was fused with Cy3 labeling and C-terminal antibody gene. The expressed fusion protein can bind to streptavidin modified fluorescent nanoparticles and detect Hantaan virus nucleoprotein on protein chip. In this study immunofluorescence protein chip mediated by single chain antibody was used to detect Hantaan virus nucleoprotein. Nuclear proteins of 10 pg/ml can be detected in a short period of time, and lower concentrations of 10 FG / ml can be detected by immune-PCR method, which are more sensitive than traditional ELISA(1 渭 g / ml methods. We have preliminarily tried to use fluorescent polymers to carry out homogenous detection of NP. Compared with other methods, it is very simple and rapid, and can detect nuclear proteins of 10 ng/ml in a few minutes.
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1

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