结核分枝杆菌反应性T细胞Elispot IL-12检测技术的建立

发布时间：2018-08-20 12:47

【摘要】：目的:建立一种用于评价结核分支杆菌感染后机体细胞免疫水平的检测技术,从而为结核病的特异性诊断、治疗及疗效监测提供一种新手段。方法:1.设计并合成结核分枝杆菌PPE36、TB10.4基因片段引物,用结核分枝杆菌(Mycobacterium tuberculosis,MTB)标准菌株(H37Rv)DNA做模板进行PCR反应扩增。将扩增产物与克隆载体连接、转化至大肠杆菌E.coli DH5a培养后,提取质粒进行鉴定和保存。2.将质粒与表达载体连接,转化培养,利用IPTG在大肠杆菌E.coli BL21(DE3)中进行诱导表达。对表达所得蛋白进行纯化,鉴定。3.以鼠抗人IL-12抗体包被PVDF膜,用生物素链霉亲和素法建立起Elispot IL-12检测技术。4.用诱导表达得到的PPE36、TB10.4蛋白刺激培养正常人及感染结核分枝杆菌患者外周血单个核细胞,对Elispot IL-12检测技术的临床应用可行性进行验证。结果:1.实验所得的结核分枝杆菌PPE36、TB10.4基因序列经测序证明与GenBank中序列一致,并成功表达出相应的蛋白。2.SDS-PAGE电泳结果显示PPE36蛋白在相对分子量为30kDa处、TB10.4在相对分子量为10kDa处各有一明显条带,与预期大小一致。3.Elispot结果显示,空白对照组及正常人组没有出现斑点,而感染结核分枝杆菌患者组及阳性对照组出现明显的斑点。结论:1.利用基因工程方法体外获取了结核分枝杆菌PPE36、TB10.4蛋白。2.经鉴定,目的蛋白大小与文献报道一致。3.初步建立了结核分枝杆菌反应性T细胞Elispot IL-12检测方法。
[Abstract]:Objective: to establish a new method for the detection of cellular immunity after Mycobacterium tuberculosis infection, so as to provide a new method for the specific diagnosis, treatment and monitoring of curative effect of tuberculosis. Method 1: 1. Primers were designed and synthesized for the gene fragment of Mycobacterium tuberculosis (PPE36) TB10.4. The PCR reaction was carried out by using Mycobacterium tuberculosism DNA (H37Rv) as template. The amplified product was ligated with the clone vector and transformed into E. coli E.coli DH5a culture. The plasmid was identified and preserved. The plasmid was connected with the expression vector and transformed into culture. The expression was induced by IPTG in Escherichia coli E.coli BL21 (DE3). The expressed protein was purified and identified. The PVDF membrane was coated with mouse anti-human IL-12 antibody and the Elispot IL-12 detection technique was established by biotin streptavidin method. The peripheral blood mononuclear cells (PBMC) of normal persons and patients infected with Mycobacterium tuberculosis were stimulated with PPE36 TB10.4 protein and the feasibility of clinical application of Elispot IL-12 detection technique was verified. The result is 1: 1. The sequence of Mycobacterium tuberculosis (PPE36) TB10.4 gene was confirmed by sequencing, and the corresponding protein was successfully expressed. 2. SDS-PAGE electrophoresis showed that the PPE36 protein had a distinct band at the relative molecular weight of 30kDa and the relative molecular weight of TB10.4 was 10kDa. The results of Elispot were consistent with the expected size. 3. The results of Elispot showed that there were no spots in the blank control group and the normal control group, but obvious spots appeared in the mycobacterium tuberculosis infection group and the positive control group. Conclusion 1. The gene engineering method was used to obtain the protein of Mycobacterium tuberculosis PPE36 TB10.4 in vitro. It was identified that the size of the target protein was consistent with that reported in the literature. A method for the detection of reactive T cell Elispot IL-12 of Mycobacterium tuberculosis was established.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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