可用于血清内抗体即时检测的高性能BRET系统的研究

Antibodies in serum play a key role in human immune response and are often related to diseases and treatments based on antibodies. Therefore, point-of-care (POC) detection of antibody not only enables rapid screening of diseases but also provides timely evaluation and guidance for antibody-based therapy. To meet this need, a POC detection system LUMABS based on bioluminescence resonance energy transfer (BRET) has been recently developed. This system consists of a BRET pair (luciferase Nluc and green fluorescent protein mNeonGreen) and an antibody-sensing module, and is becoming attractive because of easy operation, simple equipment, low cost and high sensitivity. However, the signal-to-noise ratio (SNR) and sensitivity of BRET are relatively low. In this proposal, we will engineer a bright luciferase Nluc2 and a high-performance green fluorescent protein sfmClover using structure-guided rational design and random mutagenesis, then develop a new LUMABS system with higher SNR and sensitivity, and finally detect EB-specific antibodies with the LUMABS2 system using a smart phone. This project will facilitate understanding of the relationship between crystal structures and optical properties of luminescent proteins, speed up the application of LUMABS2 in POC detection of antibodies, and also contribute to development of genetically encoded probes for other biomolecules.

血清内抗体是人体免疫反应的重要参与者，与疾病发生和抗体药物治疗密切相关。因而，血清抗体的即时检测不仅为疾病快速筛查提供可靠的方法，而且为抗体药物治疗提供及时的评估。最近，研究人员开发了基于生物发光共振能量转移（BRET）的即时检测系统LUMABS。该系统由BRET对（荧光素酶Nluc和绿色荧光蛋白mNeonGreen）和抗体感应模块组成，具有操作简便、仪器简单、成本低、灵敏度高等优点。然而，该系统在血清内的信噪比和灵敏度较差。本项目拟通过蛋白质晶体结构介导的理性设计和随机突变技术，开发高亮度Nluc2和高性能绿色荧光蛋白sfmClover；并依据BRET原理，研发高性能的LUMABS2系统；最终，结合智能手机实现EB病毒特异性抗体的即时检测。本项目研究将加深理解发光蛋白结构和光学性能之间的关系，促进LUMABS系统在血清抗体即时检测中的应用，并为研发检测其它分子的BRET探针奠定基础。

血浆内抗体检测属于复杂样本分析，对准确性和灵敏度要求高。BRET技术因为无需外部激发光源以及操作简单，能实现快速高灵敏度检测，已被广泛应用于生物医学研究和药物筛选。本项目通过优化Nluc和绿色荧光蛋白的间的生物发光共振能量转移(BRET)，构建可用于血浆抗体实时检测的高性能BRET体系，并以HIV1为模型验证该体系的优势，开发了BRET系列探针系统，用于检测血浆内如艾滋病毒、新冠病毒、登革病毒、EB病毒等多种病原体的不同抗体。该项目对完善疾病诊断、分期、疗效监测和预后判断等有重要科学意义，并有望成为临床治疗方案的依据。后续还可与公司合作开发相关试剂盒或小型检测设备，实现个体化健康医疗服务，具有广泛应用价值。