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超折叠绿色荧光蛋白纳米抗体复合体结构功能学研究摘要 绿色荧光蛋白作为基因中常用的标记蛋白，具有荧光稳定、检测方便等优点，广泛应用于生物工程领域。近年来，从骆驼体内重链抗体克隆其可变区得到的纳米抗体，相较于传统抗体具有更小的分子量，更佳的抗原结合性等。这些特性使其不仅在药物开发等医学领域有着广阔应用前景，也为蛋白结构功能学研究提供了新方法。本课题通过自主构建超折叠绿色荧光蛋白的质粒和其特异性的纳米抗体的质粒，经大肠杆菌BL21(DE3)表达后，先后采用镍亲和柱、分子筛柱对两种蛋白分别进行分离纯化，获得纯的超折叠绿色荧光蛋白和其特异性纳米抗体。采用 SDS-PAGE，Native-PAGE，Western blot等方法对两种蛋白反应复合体的性质进行探究。为接下来对复合体结晶，从分子结构上阐明相互作用机制提供了条件。41307
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毕业论文关键词 绿色荧光蛋白 纳米抗体 载体构建 蛋白表达 分离纯化
Title Structure and function studies of the superfolder green fluorescent protein-nanobody complex
Abstract Given the advantages of stable fluorescent signal, easy manipulation, green fluorescent protein (GFP) is an important marker protein which has been widely used in biotechnology, cell biology. In the past few decades, the single domain antigen-binding fragments known as “nanobodies” have many beneficial features including small size, high solubility and stability, well expressed in microorganisms. These beneficial properties enable nanobodies not only to be exploited in broad applications including the drug development, immuno-diagnosis and immumo-therapy, but also to become a promising tool for structural biology. In this study, we constructed the vector superfolder green fluorescent protein (sfGFP) and the vector of the sfGFP specific nanobody. Both proteins were expressed in E.coli, and purified through a Ni-NTA affinity column and G-75 gel filtration column, successively. SDS-PAGE, native-PAGE and Western blot methods were carried out to probe the reaction of two proteins.
Keywords green fluorescent protein nanobody vector construction protein expression protein purification
目次
1引言.1
1.1绿色荧光蛋白及其变体的结构功能学进展1
1.1.1绿色荧光蛋白简介1
1.1.2绿色荧光蛋白结构1
1.1.3绿色荧光蛋白的优点及其应用2
1.1.4超折叠绿色荧光蛋白简介3
1.2纳米抗体3
1.2.1纳米抗体简介.3
1.2.2纳米抗体结构.4
1.2.3纳米抗体的特性及应用前景4
1.3GFP与NbGFP的研究进展5
1.4课题研究内容及意义.5
2sfGFP与NbsfGFP原核表达载体构建.6
2.1实验仪器和材料6
2.2实验方法9
2.2.1sfGFP原核表达载体的构建.9
2.2.2sfGFP重组载体的验证.10
2.2.3NbsfGFP原核表达载体的构建10
2.2.4NbsfGFP重组载体的验证12
2.3结果与讨论13
2.3.1sfGFP表达载体构建结果.13
2.3.2NbsfGFP表达载体构建结果14
3sfGFP与NbsfGFP的表达纯化.16
3.1实验仪器和材料16
3.2实验方法18
3.2.1sfGFP的转化及诱导表达.18
3.2.2sfGFP的纯化分离.19
3.2.3NbsfGFP的转化及诱导表达20
3.2.4NbsfGFP的纯化分离21
3.3结果与讨论22
3.3.1sfGFP的表达纯化结果.22
3.3.2NbsfGFP的表达纯化结果23
4NbsfGFP与sfGFP复合体的相互作用.24