【摘要】：肌紅蛋白是正常存在于人體肌肉中的一種貯氧蛋白，當(dāng)肌肉損傷時，會高濃度釋放到血液中，是肌肉損傷的特異性標(biāo)志物。很多疾病與肌肉損傷有關(guān)，因此肌紅蛋白在血液中的水平往往作為一些疾病的指標(biāo)，比如急性心肌梗死、心絞痛、多發(fā)性肌炎等。傳統(tǒng)的肌紅蛋白檢測方法往往在其特異性及檢測靈敏度等方面遇到了挑戰(zhàn)，因此找到一種能高特異性高親和力識別肌紅蛋白的配體對提高肌紅蛋白檢測的特異性和靈敏度等方面有著重要的意義。核酸適體作為一種新型的配體，，具有目標(biāo)范圍廣，穩(wěn)定性好，生產(chǎn)簡單、易于修飾等特點，在很多方面甚至可以與抗體媲美，為解決上述挑戰(zhàn)帶來了機遇，因此核酸適體的篩選成為近年來研究的焦點。與此同時，微流控芯片作為一種新型的化學(xué)、生物分析檢測技術(shù)平臺得到了快速的發(fā)展。因其具有快速、高效、高通量、低成本、低樣品量、微型化、集成化等優(yōu)點，特別是在分離領(lǐng)域具有獨特的優(yōu)勢，所以越來越多地被用于核酸適體篩選方面的研究。 本論文利用不同結(jié)構(gòu)的微流控芯片，以肌紅蛋白為篩選目標(biāo)，對其特異性結(jié)合的核酸適體進行了快速篩選，并對得到的序列進行了分析和優(yōu)化： （1）基于微珠固定的微流控芯片的肌紅蛋白特異性核酸適體的篩選 通過將肌紅蛋白修飾在聚苯乙烯微珠上，并將修飾好的微珠固定于微流控芯片通道內(nèi)的一個收縮段，借助于微流控芯片在分離中的優(yōu)點，有效地將與肌紅蛋白結(jié)合的序列從整個隨機文庫中分離開來。經(jīng)過總共6輪的篩選，并對各輪次富集產(chǎn)物進行親和力的考察，選取第4輪富集產(chǎn)物進行克隆測序，最終獲得3條對肌紅蛋白親和力高、特異性好的序列。 （2）正反篩單元集成的微流控芯片用于肌紅蛋白特異性核酸適體的篩選 在第一部分工作的基礎(chǔ)上，為了進一步減少整個篩選過程的時間和獲得更適合于應(yīng)用的序列，對芯片的結(jié)構(gòu)和初始文庫進行了重新設(shè)計。通過設(shè)計具有兩個收縮段的通道，將目標(biāo)蛋白和對照蛋白修飾的微珠分別固定，在只通一遍溶液的情況下同時完成正反篩選。在文庫序列的設(shè)計中，對兩端引物序列進行了封閉，有效避免了該部分序列參與到與目標(biāo)結(jié)合時產(chǎn)生的三維結(jié)構(gòu)中。經(jīng)過8輪的篩選，并對第6、7、8輪富集產(chǎn)物的序列進行測序分析，獲得4條親和力高、特異性好的40個堿基的序列。通過對篩選出序列的二級結(jié)構(gòu)分析，將序列長度進行了優(yōu)化。
[Abstract]:Myoglobin is a kind of oxygen storage protein which normally exists in human muscle. When muscle injury, it will be released into blood at high concentration, which is a specific marker of muscle injury. Many diseases are related to muscle injury, so the level of myoglobin in the blood is often used as an indicator of some diseases, such as acute myocardial infarction, angina pectoris, polymyelitis and so on. Traditional myoglobin detection methods often face challenges in their specificity and sensitivity. Therefore, it is of great significance to find a ligand which can recognize myoglobin with high specificity and affinity in order to improve the specificity and sensitivity of myoglobin detection. Nucleic acid aptamer, as a new type of ligand, has the characteristics of wide target range, good stability, simple production, easy modification and so on. It can even be compared with antibody in many aspects, which brings opportunities to solve the above challenges. Therefore, the screening of aptamers has become the focus of research in recent years. At the same time, microfluidic chip, as a new type of chemistry, biological analysis and detection technology platform has been developed rapidly. Because of its rapid, high efficiency, high throughput, low cost, low sample size, miniaturization, integration and other advantages, especially in the field of separation, it has been more and more used in nucleic acid aptamer screening. In this paper, the specific binding aptamers of myoglobin were screened rapidly by using microfluidic chips with different structures and myoglobin as the screening target. The obtained sequences were analyzed and optimized: (1) myoglobin specific nucleic acid aptamer based on microfluidic chip fixed by beads was screened by modifying myoglobin on polystyrene beads. The modified beads were fixed in a contraction segment in the channel of the microfluidic chip. With the help of the advantages of the microfluidic chip in the separation, the sequence bound to myoglobin was effectively separated from the whole random library. After a total of 6 rounds of screening, and the affinity of each round of enrichment products was investigated, the fourth round of enrichment products were selected for cloning and sequencing, and finally three sequences with high affinity and good specificity to myoglobin were obtained. (2) the integrated microfluidic chip of positive and negative screening unit is used for the screening of myoglobin specific nucleic acid aptamers on the basis of the first part of the work, in order to further reduce the time of the whole screening process and obtain a more suitable sequence for application. The structure and initial library of the chip are redesigned. By designing a channel with two contraction segments, the target protein and the control protein modified beads were fixed respectively, and the positive and negative screening was completed at the same time when the solution was only passed through the solution. In the design of library sequence, the primer sequence at both ends is closed, which effectively avoids the participation of this part of the sequence in the three-dimensional structure produced when combined with the target. After 8 rounds of screening and sequencing analysis of the sequences of the enrichment products in the 6th and 7th rounds, four sequences with high affinity and good specificity were obtained. Through the analysis of the secondary structure of the selected sequence, the length of the sequence is optimized.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王成剛;莫志宏;;核酸適體技術(shù)研究進展[J];生物醫(yī)學(xué)工程學(xué)雜志;2006年02期

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