检测单分子蛋白个体的细胞溶素A纳米孔芯片

THE Cytolysin A Protein Nanopore For the Detection of Individual Monomer Protein

王卓飞

11930226

硕士

材料工程

李毅

2021-05-19

2021-06-18

南方科技大学

深圳

生物体内的各种酶、抗原和抗体在本质上都是蛋白质，是维持生物体运转和修复的重要成分。如何实现对功能蛋白质的快速与低成本检测来研究目标蛋白的功能是目前研究的一个重要方向。兴起于上世纪90年代的纳米孔检测技术，具有操作简单、灵敏度高、检测速度快以及无需标记等优点，为我们蛋白质分析提供了新的解决思路。他的核心原理为：当溶液中的被测物在电场力的作用下通过与其尺寸相近的纳米孔时，会使纳米孔通道的电信号发生变化，分析这些变化的电信号，有助于我们反向识别被检测物。大肠杆菌细胞溶血素A(Cytolysin A，ClyA)作为近几年被应用于纳米孔检测的一种新型生物纳米孔蛋白，孔内径大于5 nm，可直接应用于生物大分子的检测之中，但自然界存在的野生型ClyA生物纳米孔不稳定。Tn5转座子酶（Transposase, Tn5）是转座子酶中的一种，具有诱导基因突变的功能，是基因诊断学研究不可或缺的工具之一。而DNA聚合酶Phi29（Phi29 DNA polymerase, Phi29）是从噬菌体中克隆出的嗜温DNA聚合酶，DNA聚合酶Phi29在聚合酶链式反应(PCR)和生物诊断中具有不可替代的作用。本文通过大肠杆菌的异源基因表达，构建了多种ClyA突变体生物纳米孔蛋白，并选取了一种突变体对Tn5和Phi29进行了识别。具体的内容包括ClyA工程菌的培养,目的蛋白的表达和纯化，纳米孔测试实验平台的搭建以及测试数据的收集。

Proteins such as enzymes, antigens, and antibodies are all irreplaceable components in maintaining the operation and restoration of organisms. How to achieve a rapid and low-cost detection method for the target protein and study the function of target protein is currently a significant direction for medical research. The nanopore detection technology, which emerged in the 1990s，has the advantages of simple operation, high sensitivity, fast detection speed, and do not need labels, it provides us a new solution for protein detection. His key principle: when the molecule, whose dimension is comparable to the nanopore, needs to be analyzed passes through the nanopore under the action of the electrophoretic force in salt solution, the current flow of the nanopore channel will change. Analyzing these changed current signals will help us identify the object which were detected. as a new type of bio-nanopore that has been applied in nanopore detection for recent years, Cytolysin A (ClyA) has a larger inner pore diameter than 5 nm and can be directly used to the detection macromolecules, but The wild-type ClyA bio-nanopores in nature are unstable. Tn5 transposase (Transposase, Tn5) is one of the transposase enzymes. It has the function of inducing gene mutations and is one of the indispensable tools for genetic diagnostics research. Phi29 DNA polymerase (Phi29) is a mesophilic DNA polymerase cloned from bacteriophage Phi29. DNA polymerase plays an irreplaceable role in polymerase chain reaction (PCR) and biological diagnosis. In this paper, a variety of ClyA mutant biological nanopores were constructed through heterologous gene expression in Escherichia coli. A mutant of ClyA was choosen to identify Tn5 and Phi29. The content includes the cultivation, expression and purification for the ClyA mutant protein, the construction of the experimental platform, and the collection of data.

蛋白质检测 ClyA生物纳米孔 Tn5转座子酶 Phi29 DNA聚合酶

Protein Detection ClyA Protein Nanopore Tn5 Transposase Phi29 DNA Polymerase

中文

独立培养

南方科技大学

王卓飞. 检测单分子蛋白个体的细胞溶素A纳米孔芯片[D]. 深圳. 南方科技大学,2021.