基于微流控芯片的肌红蛋白特异性核酸适体的筛选
发布时间：2019-06-05 07:14
【摘要】：肌红蛋白是正常存在于人体肌肉中的一种贮氧蛋白，当肌肉损伤时，会高浓度释放到血液中，是肌肉损伤的特异性标志物。很多疾病与肌肉损伤有关，因此肌红蛋白在血液中的水平往往作为一些疾病的指标，比如急性心肌梗死、心绞痛、多发性肌炎等。传统的肌红蛋白检测方法往往在其特异性及检测灵敏度等方面遇到了挑战，因此找到一种能高特异性高亲和力识别肌红蛋白的配体对提高肌红蛋白检测的特异性和灵敏度等方面有着重要的意义。核酸适体作为一种新型的配体，，具有目标范围广，稳定性好，生产简单、易于修饰等特点，在很多方面甚至可以与抗体媲美，为解决上述挑战带来了机遇，因此核酸适体的筛选成为近年来研究的焦点。与此同时，微流控芯片作为一种新型的化学、生物分析检测技术平台得到了快速的发展。因其具有快速、高效、高通量、低成本、低样品量、微型化、集成化等优点，特别是在分离领域具有独特的优势，所以越来越多地被用于核酸适体筛选方面的研究。 本论文利用不同结构的微流控芯片，以肌红蛋白为筛选目标，对其特异性结合的核酸适体进行了快速筛选，并对得到的序列进行了分析和优化： （1）基于微珠固定的微流控芯片的肌红蛋白特异性核酸适体的筛选 通过将肌红蛋白修饰在聚苯乙烯微珠上，并将修饰好的微珠固定于微流控芯片通道内的一个收缩段，借助于微流控芯片在分离中的优点，有效地将与肌红蛋白结合的序列从整个随机文库中分离开来。经过总共6轮的筛选，并对各轮次富集产物进行亲和力的考察，选取第4轮富集产物进行克隆测序，最终获得3条对肌红蛋白亲和力高、特异性好的序列。 （2）正反筛单元集成的微流控芯片用于肌红蛋白特异性核酸适体的筛选 在第一部分工作的基础上，为了进一步减少整个筛选过程的时间和获得更适合于应用的序列，对芯片的结构和初始文库进行了重新设计。通过设计具有两个收缩段的通道，将目标蛋白和对照蛋白修饰的微珠分别固定，在只通一遍溶液的情况下同时完成正反筛选。在文库序列的设计中，对两端引物序列进行了封闭，有效避免了该部分序列参与到与目标结合时产生的三维结构中。经过8轮的筛选，并对第6、7、8轮富集产物的序列进行测序分析，获得4条亲和力高、特异性好的40个碱基的序列。通过对筛选出序列的二级结构分析，将序列长度进行了优化。
[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.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
本文编号：2493344
[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.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
【参考文献】
相关期刊论文 前1条
1 王成刚;莫志宏;;核酸适体技术研究进展[J];生物医学工程学杂志;2006年02期
本文编号：2493344
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