【摘要】：結(jié)核分枝桿菌在生長(zhǎng)的過程中會(huì)分泌多種胞外蛋白,其中CFP10和ESAT6這兩種蛋白是結(jié)核分枝桿菌分泌的特異性的蛋白,并且兩者會(huì)以一定的比例結(jié)合在一起。本論文研究利用核酸適配體檢測(cè)結(jié)核分枝桿菌分泌的融合蛋白CFP10-ESAT6。以羧基化的磁性納米粒子作為載體并加入氨基化的核酸適配體,通過熒光檢測(cè)法、SDS凝膠電泳法、互補(bǔ)鏈結(jié)合法等不同的方法確定了磁性納米粒子和適配體的比例。綠色熒光嵌入染料EVA Green在雙鏈DNA溶液中可發(fā)出強(qiáng)烈的熒光,在單鏈DNA中熒光強(qiáng)度很微弱。預(yù)先培養(yǎng)結(jié)核分枝桿菌并取適量結(jié)核分枝桿菌的菌液,加入到磁性納米粒子、dsDNA、綠色熒光染料EVA Green反應(yīng)形成的體系中,若核酸適配體和目標(biāo)物具有特異性,則已形成的雙鏈DNA會(huì)轉(zhuǎn)變?yōu)閱捂淒NA,從而利用EVA Green的熒光變化來驗(yàn)證核酸適配體和融合蛋白CFP10-ESAT6的特異性。由于蛋白質(zhì)中含有酪氨酸和色氨酸,所以蛋白質(zhì)會(huì)在280nm處出現(xiàn)紫外吸收峰,因此采用紫外吸收法對(duì)菌液中的目標(biāo)蛋白CFP10-ESAT6進(jìn)行了粗略的定量。因此這部分實(shí)驗(yàn)欲達(dá)到對(duì)結(jié)核病早期診斷的目的。聚乳酸是一種生物醫(yī)學(xué)材料,由于其具有表面多孔性及其生物兼容性,因此被廣泛應(yīng)用于藥品的負(fù)載與運(yùn)輸中。利福平是一種應(yīng)用最廣泛的抗結(jié)核藥物。這部分實(shí)驗(yàn)我們制備了聚乳酸負(fù)載利福平的微球,測(cè)得其產(chǎn)率、負(fù)載量、包封率分別為63.56%,13%,20.35%,并在后期加入了熒光物FAM修飾的核酸適配體,聚乳酸、利福平、熒光化的適配體可形成一個(gè)復(fù)合物。充分震蕩洗滌后檢測(cè)復(fù)合物的熒光特性和紫外吸收值。檢測(cè)結(jié)果顯示復(fù)合物中依舊含有很強(qiáng)的熒光,并且將紫外吸收值和利福平的標(biāo)準(zhǔn)曲線對(duì)照發(fā)現(xiàn):核酸適配體的加入使得利福平的損失率為18.46%。這表明核酸適配體的加入對(duì)聚乳酸中利福平的影響不大。這部分實(shí)驗(yàn)達(dá)到了對(duì)于結(jié)核病早期治療的目的,為臨床上結(jié)核病的治療提供了依據(jù)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.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R52;O657.3

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