核酸适配体检测CFP10-ESAT6融合蛋白的研究

发布时间：2018-12-27 08:43

【摘要】：结核分枝杆菌在生长的过程中会分泌多种胞外蛋白,其中CFP10和ESAT6这两种蛋白是结核分枝杆菌分泌的特异性的蛋白,并且两者会以一定的比例结合在一起。本论文研究利用核酸适配体检测结核分枝杆菌分泌的融合蛋白CFP10-ESAT6。以羧基化的磁性纳米粒子作为载体并加入氨基化的核酸适配体,通过荧光检测法、SDS凝胶电泳法、互补链结合法等不同的方法确定了磁性纳米粒子和适配体的比例。绿色荧光嵌入染料EVA Green在双链DNA溶液中可发出强烈的荧光,在单链DNA中荧光强度很微弱。预先培养结核分枝杆菌并取适量结核分枝杆菌的菌液,加入到磁性纳米粒子、dsDNA、绿色荧光染料EVA Green反应形成的体系中,若核酸适配体和目标物具有特异性,则已形成的双链DNA会转变为单链DNA,从而利用EVA Green的荧光变化来验证核酸适配体和融合蛋白CFP10-ESAT6的特异性。由于蛋白质中含有酪氨酸和色氨酸,所以蛋白质会在280nm处出现紫外吸收峰,因此采用紫外吸收法对菌液中的目标蛋白CFP10-ESAT6进行了粗略的定量。因此这部分实验欲达到对结核病早期诊断的目的。聚乳酸是一种生物医学材料,由于其具有表面多孔性及其生物兼容性,因此被广泛应用于药品的负载与运输中。利福平是一种应用最广泛的抗结核药物。这部分实验我们制备了聚乳酸负载利福平的微球,测得其产率、负载量、包封率分别为63.56%,13%,20.35%,并在后期加入了荧光物FAM修饰的核酸适配体,聚乳酸、利福平、荧光化的适配体可形成一个复合物。充分震荡洗涤后检测复合物的荧光特性和紫外吸收值。检测结果显示复合物中依旧含有很强的荧光,并且将紫外吸收值和利福平的标准曲线对照发现:核酸适配体的加入使得利福平的损失率为18.46%。这表明核酸适配体的加入对聚乳酸中利福平的影响不大。这部分实验达到了对于结核病早期治疗的目的,为临床上结核病的治疗提供了依据和方向。
[Abstract]:Mycobacterium tuberculosis secrete a variety of extracellular proteins including CFP10 and ESAT6 which are specific proteins secreted by Mycobacterium tuberculosis and the two proteins are bound together in a certain proportion. The purpose of this study was to detect the fusion protein CFP10-ESAT6. secreted by Mycobacterium tuberculosis by using aptamer of nucleic acid. The ratio of magnetic nanoparticles and aptamers was determined by using carboxylated magnetic nanoparticles as carriers and amino nucleic acid aptamers. The ratio of magnetic nanoparticles to aptamers was determined by fluorescence detection, SDS gel electrophoresis and complementary chain reaction. Green fluorescence embedded dye EVA Green can emit strong fluorescence in double-stranded DNA solution and weak fluorescence intensity in single-stranded DNA. Mycobacterium tuberculosis was precultured and appropriate amount of mycobacterium tuberculosis solution was added to the magnetic nanoparticles, dsDNA, green fluorescent dye EVA Green reaction system, if the nucleic acid aptamer and target have specificity, The formed double-stranded DNA will be transformed into single-stranded DNA, to verify the specificity of aptamer and fusion protein CFP10-ESAT6 by using the fluorescence changes of EVA Green. Because the protein contains tyrosine and tryptophan, the protein appears ultraviolet absorption peak at the 280nm, so the CFP10-ESAT6 of the target protein in the bacterial solution is roughly quantified by ultraviolet absorption method. Therefore, this part of the experiment to achieve the purpose of early diagnosis of tuberculosis. Polylactic acid (PLA) is a biomedical material which is widely used in drug loading and transportation due to its porous surface and biological compatibility. Rifampicin is the most widely used anti-tuberculosis drug. In this part of the experiment, we prepared the polylactic acid loaded rifampicin microspheres. The yield, loading amount and encapsulation efficiency of the microspheres were 63.56 and 13.35, respectively, and the aptamer, polylactic acid, was added with fluorescent FAM modified nucleic acid, polylactic acid, in the later stage. Rifampicin, a fluorescent aptamer, can form a complex. The fluorescence characteristics and UV absorption value of the complex were detected after full shock washing. The results showed that the complex still contained strong fluorescence, and the UV absorption value was compared with the standard curve of rifampicin. It was found that the loss rate of rifampicin was 18.46 because of the addition of nucleic acid aptamer. This indicated that the addition of aptamer of nucleic acid had little effect on rifampicin in polylactic acid. This part of the experiment has achieved the goal of early treatment of tuberculosis, and provided the basis and direction for clinical treatment of tuberculosis.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52;O657.3

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