实验海洋生物学重点实验室知识库

纳米抗体是目前已知尺寸最小的抗体，仅来源于骆驼科动物、鲨鱼等软骨鱼类，因其尺寸小，稳定性强，亲和力高，组织渗透性好等特征，在生命科学研究、体外检测和治疗性药物等领域发展迅速，应用广泛。在纳米抗体和抗原中存在一些关键氨基酸，它们在抗原抗体相互作用和抗体稳定性方面发挥重要作用，是抗体基础研究和改造应用研究关注的重点。纳米抗体中的非经典半胱氨酸即属于关键氨基酸，它们在纳米抗体中占有相当比例，多位于纳米抗体表面、识别抗原的互补决定区，且往往能够形成纳米抗体分子内的非经典二硫键。相较于传统抗体，纳米抗体分子量更小，更易进入细胞内，且对实体瘤渗透能力更强，因此在胞内抗体应用和肿瘤治疗领域独具优势。但上述应用场景都是还原环境，能够还原非经典半胱氨酸，断裂非经典二硫键，造成纳米抗体构象变化，对纳米抗体亲和力和稳定性等造成潜在影响，进而限制其在胞内和肿瘤环境中的应用。目前对于非经典二硫键的功能研究报道较少，仅有的研究报道采用半胱氨酸突变的方式研究非经典二硫键在纳米抗体中的作用，该方法无法真实模拟纳米抗体在还原环境的变化。因此，系统且深入地研究非经典半胱氨酸和非经典二硫键在纳米抗体中的功能对纳米抗体的开发应用具有重要意义。纳米抗体应用的前提是获得大量物理化学性质稳定的纳米抗体样品，目前常采用重组表达的方式制备纳米抗体，并利用重组过程中引入的额外纯化标签，对纳米抗体进行分离纯化。引入的额外的纯化标签有可能造成纳米抗体物理化学性质改变，影响纳米抗体应用。因此，如何不引入额外标签并进行纳米抗体的纯化，是纳米抗体研究的重要方向之一。BC2-tag是一个12个氨基酸形成的小肽，可与驼源纳米抗体BC2-Nb形成反向平行的β片层，并识别BC2-Nb结构域的疏水口袋。这一疏水口袋的氨基酸序列在驼源纳米抗体中较为保守。因此，在抗原抗体关键氨基酸研究的基础上，经过一定改造后，BC2-tag有望成为驼源纳米抗体通用型亲和tag，用于驼源纳米抗体重组表达后的分离纯化，避免目前纯化方法中引入的额外纯化标签，及其可能造成的纳米抗体物理化学性质变化。

此外，纳米抗体在多种生物成像技术中均能发挥提高图像分辨率的作用，但在溶液扫描隧道显微镜（溶液STM）中尚未有相关报道。不同于传统扫描隧道显微镜的低温、高真空测量环境，溶液STM能够在溶液环境中对真实生理状态的生物样品进行单分子成像。透明质酸合成酶是一种双功能酶，能够合成并跨膜转运透明质酸，目前其酶促动力学研究主要是基于传统生物化学实验的系综研究，其中关键的动态过程由于系综平均而难以观测。利用溶液STM，在接近真实生理条件下的溶液环境中，开展单分子成像研究能够有效弥补这一不足，但是透明质酸合成酶分子尺寸较小且为球形蛋白，没有明确的形貌特征，在溶液STM成像过程和图像分析过程中，如何确认透明质酸合成酶是关键难题。实验室前期获得了透明质酸合成酶特异性纳米抗体，具有nM级别的高亲和力，形成的纳米抗体和透明质酸合成酶复合物，能够为透明质酸合成酶提供较突出的形貌特征，有望辅助开展单分子层次的透明质酸合成酶的酶促动力学研究，更加精细地阐释透明质酸合成酶合成透明质酸的动态过程。

本论文选择能识别BC2-tag的驼源纳米抗体BC2-Nb和抗溶菌酶的鲨鱼源纳米抗体lyso-VNAR作为研究对象探讨非经典半胱氨酸和非经典二硫键在纳米抗体中的功能。分别使用对两个非经典半胱氨酸进行定点突变和饱和突变的方式构建BC2-Nb和lyso-VNAR的突变体，另外采用还原剂处理的方式破获BC2-Nb和lyso-VNAR的非经典二硫键，研究非经典半胱氨酸和非经典二硫键对于纳米抗体亲和力和稳定性的影响。结果表明，在亲和力方面，还原状态的BC2-Nb和lyso-VNAR结合抗原能力不受影响，而突变非经典半胱氨酸后，纳米抗体亲和力明显减弱，相较于野生型，lyso-VNAR突变体平衡解离常数数值升高13-211倍，BC2-Nb突变体则不再结合抗原。在稳定性方面，还原剂处理组和非经典半胱氨酸突变体纳米抗体的热稳定性Tm和化学稳定性Cm数值都明显降低，且下降程度基本相同。论文进一步用分子动力学模拟研究了突变后纳米抗体的构象变化，结果显示，与野生型和还原状态相比，BC2-Nb突变体整体柔性增大；而在lyso-VNAR系统中，突变非经典半胱氨酸后的互补决定区可能发生侧向滑动，进而影响该区域氨基酸与抗原的相互作用，造成亲和力下降。

在BC2-tag与BC2-Nb相互作用关键氨基酸的研究中，基于对BC2-tag与BC2-Nb复合物晶体结构的分析和分子对接模拟，本论文设计了两种BC2-tag改造策略: 一是突变与疏水口袋直接相互作用的关键氨基酸，使相应氨基酸侧链空间与纳米抗体的疏水口袋更适配，增强亲和力；二是尝试在BC2-tag C端增加能与纳米抗体序列中保守氨基酸发生相互作用的数个氨基酸。但以上两种方案的改造均未成功，这提示可能蛋白质晶体结构显示的抗原抗体相互作用方式与真实溶液状态下的不尽相同，因此依据复合物晶体结构进行的改造设计难以在溶液条件下的互作中得到验证。在利用纳米抗体辅助溶液STM生物成像研究中，本论文以透明质酸合成酶为研究对象，开展了单分子酶促动力学研究，在溶液状态下对单分子透明质酸合成酶及其合成透明质酸的动态过程分别进行溶液STM成像。透明质酸合成酶成像实验显示，单个酶分子的形貌特征不明显，加入纳米抗体后由于成像体系稳定性不足，也未能提高成像质量，扫描成像条件还需要进一步优化。

综上所述，本论文首先进行了纳米抗体和抗原相互作用关键氨基酸的研究，得到以下结论：1、纳米抗体非经典半胱氨酸的功能不仅以形成非经典二硫键的形式体现，半胱氨酸残基自身也能够参与抗原识别；此类情况下，突变非经典半胱氨酸不仅能够通过打破非经典二硫键降低纳米抗体稳定性，还能够减弱甚至丢失纳米抗体与抗原的结合能力，因此，非经典半胱氨酸突变的实验方法不能够等同于非经典二硫键破坏的方法。2、通过对BC2-tag与BC2-Nb互作关键氨基酸的改造，构建通用型驼源纳米抗体亲和tag的研究未获得正面结果，提示纳米抗体和抗原复合物晶体结构展示的相互作用方式或许与溶液状态复合物有所不同，对纳米抗体与抗原识别中的关键氨基酸的改造可能需要溶液中复合物结构数据作为更有效的改造起点。其次，本论文尝试拓展纳米抗体在单分子生物成像方向的应用，利用纳米抗体结合透明质酸合成酶以突出其形貌特征、辅助开展基于溶液STM的、单分子水平的透明质酸合成酶成像和酶促动力学研究，此前未见此类报道，是有价值的探索性实验和研究方向，但目前的实验没有取得成功，需要对成像实验体系做更多摸索和针对性的优化才有望满足实验设计目的。

Nanobodies, the smallest antibodies derived from camelids and sharks, are increasingly used in life sciences, diagnostics, and drug development due to their small size, stability, strong affinity, and excellent tissue penetration. Non-canonical cysteines, which form non-canonical disulfide bonds, play a key role in nanobody structure and function. These cysteines are often located in the complementary determining regions (CDRs) and contribute to the nanobody’s stability and antigen recognition. Compared to traditional antibodies, nanobodies have a smaller molecular weight, are more easily internalized by cells, and can penetrate solid tumors more effectively, making them promising candidates for intrabody and cancer therapies. However, these applications often occur in reducing environments, where non-canonical cysteines may be reduced, disrupting disulfide bonds and causing conformational changes in the nanobody. Such changes can affect its affinity, stability, and overall function, potentially reducing its therapeutic effectiveness. Research on the role of non-canonical disulfide bonds in nanobodies is limited, only relying on cysteine mutations to assess their impact. However, these mutations do not fully capture the behavior of non-canonical disulfide bonds under reducing conditions. A more systematic investigation of these bonds is necessary to optimize nanobody development for therapeutic applications. Nanobody applications require high-yield production, often involving purification tags to aid separation. However, these tags can alter the physicochemical properties of nanobodies, impacting their function. Therefore, one of the important directions in nanobody research is to purify nanobodies without introducing additional tags. The BC2-tag is a small peptide composed of 12 amino acids that forms an anti-parallel β-sheet with the camelid-derived nanobody BC2-Nb and recognizes a hydrophobic pocket in the BC2-Nb. This hydrophobic pocket's amino acid sequence is relatively conserved in camelid-derived nanobodies. Based on the study of key antigen-antibody residues, after certain modifications, the BC2-tag is expected to become a universal affinity tag for camelid-derived nanobodies, enabling their separation and purification after recombinant expression without introducing additional purification tags. This approach would avoid the potential alterations in the physicochemical properties of nanobodies caused by current purification methods.

Nanobodies can improve image resolution in various biological imaging techniques, but their application in Scanning Tunneling Microscopy (STM) has not been reported. Unlike traditional STM, which requires low temperature and high vacuum, liquid-phase STM allows single-molecule imaging of biological samples in physiological solutions. Hyaluronan synthase (HAS) is a bifunctional enzyme that synthesizes and translocates hyaluronan across membranes. While its enzymatic kinetics have been studied through ensemble experiments, these methods average out key dynamic processes, making them difficult to observe. Liquid-phase STM enables single-molecule imaging, overcoming this limitation. However, the small size and lack of distinct morphological features of HAS make it challenging to identify during imaging. We have developed specific nanobodies that, when complexed with HAS, generate unique morphological markers, facilitating single-molecule kinetic studies and providing deeper insights into hyaluronan synthesis.

We selected the camelid-derived nanobody BC2-Nb recognizing protein tag BC2-tag and the shark-derived nanobody lyso-VNAR against lysozyme, and constructed cysteine mutants of BC-Nb and lyso-VNAR by site-directed and saturation mutagenesis. We also treated the nanobodies with reducing agents to break non-canonical disulfide bonds and explored their effects on the affinity and stability of nanobodies. The binding ability of BC2-Nb and lyso-VNAR to their antigens remains unaffected in their reduced states. However, mutation of the non-canonical cysteines significantly impaired affinity, while the affinity of lyso-VNAR mutants decreased by 13-211-fold, and BC2-Nb mutant did not bind to the antigen at all. In terms of stability, both reducing agent treatment and cysteine mutation led to a decrease in the thermal stability and chemical stability of nanobodies. Molecular dynamics simulation experiments showed that the flexibility of BC2-Nb mutant increased compared to wild type and reduced state. In the lyso-VNAR system, the CDR3 loop of mutants may undergo lateral sliding, thereby affecting the interaction between amino acids in this region and the antigen.

For key residues involved in the BC2-tag/BC2-Nb interaction, two modification strategies were designed based on crystal structure analysis and molecular docking simulations of the BC2-tag/BC2-Nb complex. The first strategy involved mutating key residues that interact with the nanobody's hydrophobic pocket to improve fit and enhance affinity. The second strategy added residues to the C-terminus of BC2-tag to engage conserved residues in the nanobody. However, neither strategy was successful, suggesting that the antigen-antibody interactions observed in the crystal structure may differ from those in solution, highlighting the difficulty of translating structural modifications from crystal to solution phase.

In the application of nanobodies for liquid-phase STM bioimaging, this dissertation focused on hyaluronan synthase as the model enzyme for single-molecule enzymatic kinetics. Dynamic imaging of the enzyme and its synthesis of hyaluronan was performed using liquid-phase STM. The imaging of hyaluronan synthase showed that the individual enzyme molecules did not exhibit clear morphological features. Despite adding nanobodies, the imaging quality was not improved due to instability in the imaging system, indicating the need for further optimization of scanning conditions.

This dissertation investigates key residues involved in the interaction between nanobodies and their antigens, leading to the following conclusions: 1) Non-canonical cysteines in nanobodies do more than form non-canonical disulfide bonds; they can also participate in antigen recognition. Mutating these cysteines not only disrupts disulfide bond formation, reducing nanobody stability, but also weakens or abolishes antigen binding. Therefore, mutations of non-canonical cysteines cannot be equated with methods that disrupt disulfide bonds. 2) Attempts to engineer key residues in the interaction between BC2-tag and BC2-Nb to create a universal camelid nanobody affinity tag were unsuccessful. This suggests that the nanobody-antigen interaction in solution may differ from the crystal structure, highlighting the need for solution-phase structural data when engineering nanobody-antigen recognition. Additionally, this dissertation explores the use of nanobodies in single-molecule bioimaging by binding them to hyaluronan synthase to enhance its morphological features. The aim was to conduct single-molecule imaging and enzymatic kinetics studies of hyaluronan synthase using liquid-phase STM. While this is an innovative approach with no prior reports, the current experiments have not been successful, requiring further optimization and system development.

第1章 绪论 1

1.1 纳米抗体简介 1

1.1.1 纳米抗体的发现 1

1.1.2 纳米抗体结构特征和功能特性 2

1.2 纳米抗体的重组表达 4

1.3 纳米抗体应用 5

1.4 纳米抗体中的非经典半胱氨酸及非经典二硫键 7

1.5 纳米抗体BC2-Nb与小肽BC2-tag 10

1.6 研究意义与目标 10

第2章 驼源纳米抗体BC2-Nb非经典半胱氨酸和非经典二硫键的功能研究 13

2.1 实验方法 13

2.1.1 抗原GFP-BC2-tag的制备 13

2.1.2 纳米抗体BC2-Nb的制备 16

2.1.3 非经典半胱氨酸突变体BC2-Nb_{C55A_C110S}的制备 18

2.1.4 表面等离子共振技术（SPR）研究非经典半胱氨酸和非经典二硫键对BC2-Nb亲和力的影响 18

2.1.5 分子排阻层析共纯化研究非经典半胱氨酸和非经典二硫键对BC2-Nb结合抗原的影响 19

2.1.6 分子动力学模拟研究非经典二硫键缺失后BC2-Nb的结构变化 20

2.1.7 非经典二硫键对BC2-Nb稳定性影响 20

2.2 实验结果与讨论 21

2.2.1 抗原GFP-BC2-tag的纯化结果 21

2.2.2 纳米抗体BC2-Nb和突变体BC2-Nb_{C55A_C110S}的纯化结果 23

2.2.3 非经典半胱氨酸和非经典二硫键对BC2-Nb亲和力的影响 25

2.2.4 BC2-Nb与抗原GFP-BC2-tag的分子排阻层析共纯化结果 27

2.2.5 BC2-Nb分子动力学模拟研究结果 30

2.2.6 非经典二硫键对BC2-Nb稳定性影响 34

第3章 鲨鱼源纳米抗体lyso-VNAR非经典半胱氨酸和非经典二硫键功能研究 39

3.1 实验方法 39

3.1.1 抗原lysozyme制备 39

3.1.2 抗原lysozyme还原剂耐受实验 39

3.1.3 纳米抗体lyso-VNAR的制备 40

3.1.4 纳米抗体lyso-VNAR非经典半胱氨酸饱和突变文库的构建和筛选 43

3.1.5 高亲和力lyso-VNAR非经典半胱氨酸突变体的制备 47

3.1.6 质谱鉴定lyso-VNAR非经典二硫键的形成与断裂 48

3.1.7 表面等离子共振技术研究非经典半胱氨酸和非经典二硫键对lyso-VNAR亲和力的影响 49

3.1.8 分子排阻层析共纯化研究非经典半胱氨酸和非经典二硫键对lyso-VNAR结合抗原的影响 49

3.1.9 lyso-VNAR非经典半胱氨酸突变体与抗原共结晶 50

3.1.10 分子动力学模拟研究非经典二硫键缺失后lyso-VNAR的结构变化 51

3.1.11 非经典二硫键对lyso-VNAR稳定性影响 51

3.2 实验结果与讨论 52

3.2.1 抗原lysozyme纯化结果 52

3.2.2 还原环境lysozyme酶活性测定结果 53

3.2.3 lyso-VNAR表达纯化结果 54

3.2.4 lyso-VNAR非经典半胱氨酸饱和突变文库构建和筛选结果 56

3.2.5 lyso-VNAR非经典二硫键形成与断裂的质谱鉴定结果 59

3.2.6 非经典半胱氨酸和非经典二硫键对lyso-VNAR亲和力的影响 60

3.2.7 lyso-VNAR与抗原lysozyme的分子排阻层析共纯化结果 63

3.2.8 lyso-VNAR非经典半胱氨酸突变体与抗原共结晶结果 64

3.2.9 lyso-VNAR分子动力学模拟研究结果 67

3.2.10 非经典二硫键对lyso-VNAR稳定性影响 70

第4章 BC2-tag改造为驼源纳米抗体通用型亲和tag的研究 75

4.1 实验方法 75

4.1.1 驼源纳米抗体的表达纯化 75

4.1.2 BC2-tag与驼源纳米抗体分子排阻层析共纯化 75

4.1.3 聚焦纳米抗体疏水口袋的BC2-tag关键氨基酸的突变改造 76

4.1.4 聚焦增加纳米抗体相互作用氨基酸位点的BC2-tag关键氨基酸的突变改造 76

4.2 实验结果与讨论 77

4.2.1 驼源纳米抗体氨基酸序列比对结果 77

4.2.2 BC2-tag与驼源纳米抗体分子排阻层析共纯化结果 78

4.2.3 聚焦纳米抗体疏水口袋的BC2-tag突变体与驼源纳米抗体分子排阻层析共纯化结果 79

4.2.4 聚焦增加纳米抗体相互作用氨基酸位点的BC2-tag突变体与驼源纳米抗体分子排阻层析共纯化实验结果 83

第5章 纳米抗体辅助溶液STM研究透明质酸合成酶的单分子酶促动力学 87

5.1 研究背景 87

5.1.1 生物研究单分子技术简介 87

5.1.2 溶液STM简介 89

5.1.3 透明质酸简介 91

5.1.4 透明质酸合成酶简介 92

5.1.5 膜蛋白和类膜体系简介 94

5.1.6 研究意义与目标 96

5.2 实验方法 98

5.2.1 透明质酸合成酶的制备 98

5.2.2 透明质酸合成酶的Nanodisc组装 99

5.2.3 透明质酸合成酶活性检测 100

5.2.4 透明质酸合成酶溶液STM单分子酶促动力学研究 101

5.3 结果与讨论 103

5.3.1 透明质酸合成酶和失活突变体纯化结果 103

5.3.2 透明质酸合成酶的Nanodisc组装结果 104

5.3.3 透明质酸合成酶活性检测实验结果 105

5.3.4 透明质酸成像结果 110

5.3.5 空白溶液对照组溶液STM成像结果 111

5.3.6 透明质酸合成酶失活突变体溶液STM成像结果 112

5.3.7 anti-His抗体溶液STM成像结果 113

5.3.8 透明质酸合成酶合成透明质酸动态过程的溶液STM成像结果 114

5.3.9 纳米抗体辅助透明质酸合成酶合成透明质酸动态过程的溶液STM成像结果 117

第6章 总结与展望 121

6.1 BC2-Nb非经典半胱氨酸与非经典二硫键功能研究总结与展望 121

6.2 lyso-VNAR非经典半胱氨酸与非经典二硫键功能研究总结与展望 121

6.3 纳米抗体通用型亲和tag BC2-tag改造研究总结与展望 123

6.4 纳米抗体辅助溶液STM生物成像研究总结与展望 123

参考文献 125

致谢 139

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 141