实验海洋生物学重点实验室知识库

单域抗体是目前发现的最小的有抗原结合功能的蛋白质片段，在疾病诊断、疾病治疗、科学研究等方面具有广泛应用。目前已知的单域抗体包括来源于骆驼科动物的VHH和来源于软骨鱼类的VNAR。其中，对VHH的研究已非常广泛，但对VNAR的研究较匮乏。在单域抗体研究中，主要以水溶性蛋白作为抗原开展研究，较少以膜蛋白作为抗原。在已完成的基因测序中，约30%的基因编码膜蛋白，这些膜蛋白在信号转导和物质转运过程中发挥重要生理功能。单域抗体能够锁定膜蛋白的特定构象，在膜蛋白的结构与功能研究中发挥独特作用。因此，以膜蛋白为抗原开展鲨鱼源单域抗体的相关研究具有重要价值。本研究以透明质酸合成酶为抗原，筛选了条纹斑竹鲨来源的单域抗体，并对其进行了表达纯化和功能分析。

本论文的研究内容及结果包括：

1）利用大肠杆菌表达系统异源表达纯化了透明质酸合成酶，并利用纸层析技术结合同位素标记技术测定其活性。实验结果显示，本研究中表达纯化的透明质酸合成酶纯度较高、稳定性好、具有活性。

2）将透明质酸合成酶重组至脂质体，分六次注射至条纹斑竹鲨体内，以诱导并加强其免疫反应，用免疫后的条纹斑竹鲨淋巴细胞总RNA构建了单域抗体免疫文库。用噬菌体表面展示技术从上述免疫文库中淘选了透明质酸合成酶特异性的单域抗体（三次迭代的淘选过程）。最终利用噬菌体展示酶联免疫吸附技术获得了8个透明质酸合成酶特异性的单域抗体序列。这8个序列间，除CDR3区的氨基酸序列存在较大差异外，其余部分序列相似性极高。

3）在大肠杆菌细胞质中表达了透明质酸合成酶特异性的单域抗体，并利用分子排阻层析实验、等温滴定量热实验、核磁共振滴定实验、透明质酸合成酶活性测定实验分别探究了单域抗体与透明质酸合成酶的相互作用。其中，等温滴定量热实验和核磁共振滴定实验结果均直接表明，表达纯化获得的单域抗体VNAR1能够与透明质酸合成酶结合，等温滴定量热实验测得的两者间的平衡解离常数约为171 µM。同时，透明质酸合成酶活性检测实验结果也间接证明了两者间具有相互作用。

总之，本研究筛选并制备了透明质酸合成酶特异性的鲨鱼源单域抗体，并对其进行了功能分析，这些工作丰富了针对膜蛋白抗原的鲨鱼源单域抗体的筛选与制备研究，拓宽了鲨鱼源单域抗体的应用范围，并为所在实验室系统开展鲨鱼源单域抗体的研究奠定了基础。

As the smallest functional antigen-binding fragment, single domain antibodies are widely used in disease diagnosis, disease treatment, and scientific research. Single domain antibodies include VHH derived from camelid animals and VNAR from cartilaginous fish. In this field，many researchers focus on VHH, but few on VNAR. Soluble proteins are mainly used as antigen in the field of single domain antibodies, but membrane proteins are rarely used. About 30% sequenced genes, encode membrane proteins, which play important physiological role in signal transduction and transmembrane transport. Single domain antibodies can lock the specific conformation of membrane proteins and play a unique role in the study of the structure and function of membrane proteins. Therefore, it is of great value to use membrane protein as antigens in the study of shark-derived single domain antibodies. In this study, hyaluronan synthase was used as antigen to screen single domain antibodies from Chiloscyllium plagiosum. The expression, purification and functional evaluation of these single domain antibodies was included in this thesis as well.
This research mainly contains: 
1) The hyaluronan synthase was expressed in E.coli and purified by affinity chromatography and size-exclusion chromatography. Then paper chromatography combined with isotope labeling technology were used to determine the activity of purified hyaluronan synthase. The purified protein in this study was of high purity, good stability and was in active state.
2) The hyaluronan synthase was reconstituted into liposome, and then injected into Chiloscyllium plagiosum to induce its immune response. A single domain antibodies immune library was constructed using the total RNA of lymphocytes from the immunized Chiloscyllium plagiosum, followed by phage display to pan the specific single domain antibodies from the above-mentioned immune library (three iterations of panning process). Finally, eight hyaluronan synthase specific single domain antibodies were obtained using phage display enzyme-linked immunosorbent technology. The sequences in these single domain antibodies were highly similar, except for the CDR3 region.
3) Single domain antibodies specific for hyaluronan synthase were expressed and purified in the cytoplasm of E. coli, the affinity between hyaluronan synthase and single domain antibodies was determined using size-exclusion chromatography, isothermal titration calorimetric, nuclear magnetic resonance titration experiment and hyaluronan synthase activity experiment. Among them, the results of isothermal titration calorimetry and nuclear magnetic resonance titration experiment directly show that VNAR1 could bind to hyaluronan synthase, and the dissociation constant measured by isothermal titration calorimetry is about 171 µM. The increasement of hyaluronan synthase activity by adding single domain antibodies also indirectly support the interaction between hyaluronan synthase and VNAR1.
In summary, this study screened and prepared shark-derived single domain antibodies specific for hyaluronan synthase and performed functional analysis. This project enriches the screening and preparation of shark-derived single domain antibodies against membrane protein antigens. It broadens the application of shark-derived single domain antibodies, and lays the foundation for the laboratory to carry out the research of shark-derived single domain antibodies.

第1章 绪论    1
1.1 传统抗体及其衍生物简介    1
1.1.1 传统抗体简介    1
1.1.2 传统抗体衍生物简介    2
1.2 单域抗体简介    2
1.2.1 单域抗体的结构分析    3
1.2.2 单域抗体的特性    5
1.2.3 单域抗体的开发利用    8
1.3 单域抗体的筛选与制备    11
1.3.1 单域抗体筛选方案    11
1.3.2 单域抗体制备方案    15
1.4 透明质酸及透明质酸合成酶简介    16
1.5 选题背景、研究目标与研究内容    17
1.5.1 选题背景    17
1.5.2 研究目标    18
1.5.3 研究内容简介    18
第2章 透明质酸合成酶的表达纯化及活性验证    20
2.1 材料与方法    20
2.1.1 实验材料    20
2.1.2 实验步骤    22
2.2 实验结果    25
2.2.1透明质酸合成酶的表达纯化    25
2.2.2透明质酸合成酶的活性验证    26
2.3 结果讨论    28
第3章 透明质酸合成酶特异性的条纹斑竹鲨单域抗体筛选    29
3.1 材料与方法    29
3.1.1 实验材料    29
3.1.2 实验步骤    30
3.2 实验结果    37
3.2.1 免疫文库构建    37
3.2.2 单域抗体的氨基酸序列    37
3.3 结果讨论    38
第4章 透明质酸合成酶特异性的条纹斑竹鲨单域抗体制备    40
4.1 材料与方法    40
4.1.1 实验材料    40
4.1.2 实验步骤    42
4.2 实验结果    46
4.2.1 表达载体的构建    46
4.2.2 单域抗体的制备    46
4.3 结果讨论    47
第5章 透明质酸合成酶特异性的条纹斑竹鲨单域抗体功能评价    49
5.1 材料与方法    49
5.1.1 实验材料    49
5.1.2 实验步骤    50
5.2 实验结果    52
5.2.1 单域抗体与透明质酸合成酶的共纯化    52
5.2.2 等温滴定量热实验    53
5.2.3 核磁共振滴定实验    54
5.2.4 单域抗体对透明质酸合成酶活性的影响    56
5.3 结果讨论    56
第6章 总结和展望    61
6.1 总结    61
6.2 展望    61
参考文献    63
致 谢    75
作者简历及攻读学位期间发表的学术论文与研究成果    77