烟草蚀纹病毒蛋白酶调控的人工变构酶的初步设计
毕业论文关键词：翻译后调控；β-半乳糖苷酶；级联反应；放大器
The Preliminary Design of TEV Protease-Regulated Artificial Allosteric Enzyme
Abstract: One important purpose of synthetic biology is to develop molecular tools that can be accurately
quantified at multiple levels to achieve rational design of life system. At present, the engineering research at
the protein level has provided a variety of ideas and methods for the design of the post-translation level
regulating parts. By semi-rational design and transformation, we constructed and obtained one version of
modified β-galactosidase, which is catalyzed by the tobacco etch virus protease, and by using the difference
of catalytic activity between rearrangement fusion and fragment complementation, which is determined by
the natural properties of protein, we constructed a positive regulatory circuit at the post-translational level.
At the same time, the quantitative property analysis of the modified protein version was carried out, and the
methods of original quantitative assay were optimized in order to facilitate high-throughput experimental
operation. Finally, we established a mathematical model for the double enzyme cascade system, and the
amplification effect of the system was also explored under the guidance of the mathematical model. We
expect our post-translational regulation part could play an important role in the application practice as an
amplifier device, such as pathogen diagnosis and detection.
Key Words: post-translational regulation; β-galactosidase; cascade reaction; amplifier
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法3
1.1 主要实验材料 3
1.1.1 菌株与质粒 3
1.1.2 培养基与基础试剂配制3
1.1.3 邻硝基苯 β-D-半乳吡喃糖苷 （ONPG） 定量实验相关试剂配制4
1.2 主要实验仪器4
1.3 基础分子生物学实验方法4
1.3.1 大肠埃希氏菌化学感受态细胞的制备4
1.3.2 质粒或连接体系的热激法转化5
1.3.3 多聚酶链式反应（PCR）获取目的片段 5
1.3.4 Gibson Assembly进行目标载体构建 5
1.3.5 使用商业试剂盒进行质粒小提、PCR 产物纯化及DNA切胶回收 5
1.4 主要测试实验方法 7
1.4.1 TEV 蛋白酶体内活性测试 7
1.4.2 X-gal 染色法进行 β-Gal 定性验证 7
1.4.3 ONPG实验法进行 β-Gal 定量验证 7
2 结果与分析 7
2.1 测试用底盘菌株的选取与TEV蛋白酶表达系统的构建与优化 7
2.2 对 β-半乳糖苷酶的半理性变构设计与结果分析 10
2.2.1 基于 α-互补原理的简单重排设计 10
2.2.2 基于简单重排的系统删减设计 11
2.2.3 在 β-Gal 全长范围选取多位点进行半理性拆分设计 14
2.3 利用 ONPG进行定量实验 16
2.3.1 “β-ψ”拆分的 ONPG 定量测试 16