SPA-IgG复合体分子内的蛋白质多靶标并行检测

In this proposal, we present the first description of an innovative approach for parallel detection of multi-target proteins within a macromolecular complex based on 3D (three-dimensional) antibody probes which are stacked on solid matrix surface layer-by-layer. It mainly imitates the molecular mechanisms of immunomodulatory interference by Staphylococcus aures, being also involved in recombinant protein technology. Firstly, two anti-parallel HisTag-SPA (polyhistidine-tagged Staphylococcal protein A) fusion proteins, obtained from differentiated introduction of HisTag to SPA, are used for the assembly of five different IgG (Immunoglobulin G) antibodies one by one. Consequently, the SPA-IgG complex, which is immobilized following the chelation of HisTag with Nickel(Ⅱ), is built up. Coupled with corresponding fluorescently labeled secondary antibodies, five different target protein antigens are captured as a double antibody "sandwich" immune complex which ultimately can be imaged by using single-molecule TIRFM (total internal reflection fluorescence microscopy). Pull-down, crystal XRD (X-ray diffraction), QCM (quartz crystal microbalance) and ELISA (enzyme-linked immunosorbent assay) or other techniques, combined with molecular dynamics simulation, are used as a tool to carry out all-round, multi-angle analysis on crucial issues such as molecular mechanism of the interaction of SPA fusion protein with IgG antibody, spatial and temporal variation of multi-layer assembly, 3D orientation of functional sites and the associated immune response. And as a result, it will help us ascertain the interfacial biological processes of protein complex, structure-activity relationships, and track its mechanism for seeking feasibility of intervention. The related work products can be applied to the immobilization or identification of multi-target macromolecules, functional gene analysis, early diagnosis of serious disease,etc., and may offer a unique idea for artificial interference with the dynamic behaviors of proteins or their complexes, intended design of asymmetric molecular devices, and other foundational researches.

通过模仿葡萄球菌干扰免疫调理的分子机制，结合蛋白质重组技术，在固相基体表面构建层层堆积的三维抗体探针，首次提出在单个复合体分子内实现蛋白质多靶标并行检测的新方法。首先在SPA两端非对称引入HisTag组氨酸标签，以反向平行的方式逐层组装5种IgG抗体，SPA-IgG复合体经镍离子螯合作用固相化。再配以相应的荧光标记二抗，与5种蛋白质抗原靶标形成双抗体夹心免疫复合物，最终对其进行全内反射单分子荧光成像。拟采用Pull-down、XRD、QCM及ELISA等手段，结合分子动力学模拟全方位分析SPA与IgG的相互作用机制、层层组装的时空规律、功能位点的空间取向及与之关联的免疫响应，由此探明蛋白质复合体的界面生物过程、构效关系及其干预路径。相关工作将为多指标生物大分子的固定与识别、功能性基因分析、重大疾病的早期诊断、蛋白质及其复合体动态行为的人工干预、不对称分子器件的设计等基础研究提供一条新思路。

本课题通过模仿葡萄球菌干扰免疫调理的分子机制，结合蛋白质重组技术，在固相基体表面构建层层堆积的三维抗体探针，在复合体分子内实现蛋白质多靶标并行检测。本项目取得的主要成果有：1、在葡萄球菌蛋白A（SPA）两端非对称引入HisTag组氨酸标签肽，获得2分子HisTag-SPA，后者以反向平行的方式组装5分子IgG抗体。通过HisTag组氨酸标签与镍离子的螯合作用，将抗体以三维立体排布的方式吸附于固相基体表面，抗原受体位点进而得以充分暴露，对分析物的响应信号也由此显著增强，检测灵敏度增大了64倍以上。Pull-down实验证明了IgG抗体只能经SPA的连接间接地结合至镍离子基体，位于SPA一端的组氨酸标签与镍离子结合，而5个同源结构域结合IgG抗体的Fc段。以积分光密度（IOD）定量表示的5个杂交带的信号强度随SPA所含IgG结合域的个数逐一递增（IOD值分别为：14.55、35.78、81.53、217.37、263.15），说明经SPA结合的IgG抗体数量也逐渐增加。2、将HisTag-SPA吸附到镍离子琼脂糖凝胶珠表面，通过HisTag标签肽与镍离子之间的螯合作用将抗体进行3D固化，使抗体特异性结合抗原的活性位点充分暴露，进而结合不同的抗原分子和荧光标记二抗。利用荧光显微镜检测激发出的不同波长的荧光，同步检测抗原与抗体的特异性免疫反应，以实现蛋白质多靶标并行检测的目的。3、用三种不同的修饰方法（硅烷化、SPA固定、硅烷化加SPA固定）对镀金的石英晶片进行表面改性，利用石英晶体微天平和原子力显微镜对蛋白质在晶片表面的吸附行为进行表征，通过蛋白质分子吸附所引起的晶体谐振频率变化实时监测不同抗体与对应抗原发生的特异性免疫反应。AFM结果显示：经SPA固定的蛋白质能有效避免分子成团与位点遮蔽，蛋白质分子在晶片表面粗糙度均匀，平均高度为80 nm。项目研究成果已发表SCI论文19篇，申请国家发明专利2项。相关工作将为多指标生物大分子的固定与识别、功能性基因分析、重大疾病的早期诊断、蛋白质及其复合体动态行为的人工干预、不对称分子器件的设计等基础研究提供技术支撑与新思路，并具有良好的产业化前景。