类病毒颗粒在分离纯化过程中组装机制解析

Virus-like particles (VLPs) technique represents the development direction of novel vaccine. It is important for infectious and serious diseases prophylaxis and therapy. VLPs are usually composed of multiple subunits conjugated by non-covalent bonds or dissociable disulfide bonds easily cause dissociation or aggregation of subunits, consequently loss activity of VLPs. Activity recovery of many VLP products is less than 20%, even lower than 5%, after separation and purification. Using hepatitis B surface antigen as a model protein, this project will explore feasible methods to improve the productivity and activity of VLPs. Our study will focus on the influence of surfactant, solution environment and solid-liquid interface on the structure change of VLPs. Utilize modern advanced analysis techniques, such as differential scanning calorimeter and isothermal titration calorimeter, to investigate kinetic and thermodynamic parameters of VLPs’ conformational change. Take advantage of quartz crystal microbalance and atomic force microscopy to in-suite measure the adsorption kinetics of VLPs. Apply Zetasizer Nano ZSP system for in-suit detection of molecular weight, hydrodynamic radius and surface charge of VLPs in different solution environment and chromatography media. The assembly mechanism of VLPs during separation and purification process will be elucidated, and strategy for improving the stability of VLP structure and activity will be proposed as well. . This project will establish fundamental theory for high efficient separation and purification of VLPs.

类病毒颗粒(virus-like particles, VLPs)技术是现代新型疫苗的发展方向，在传染病和重大疾病的防治中具有重要应用前景。VLP通常含有多个亚基，亚基间由非共价键或易解离的二硫键连接，很容易在分离纯化过程中发生解聚或聚集，导致活性丧失，许多产品的活性收率不到20%甚至5%。本项目针对此问题，拟以乙型肝炎病毒表面抗原(HBsAg)为模型，研究表面活性剂、溶液环境和固液界面对VLP结构的影响。通过先进的分析技术如差示扫描量热和等温滴定量热技术研究VLP结构变化的动力学和热力学参数，石英晶体微天平联用原子力显微镜原位检测类VLP在层析介质上的吸附动力学，利用动态光散射粒度仪原位检测不同溶液环境和层析介质条件下VLP组装结构的分子量、水力学半径和表面电荷的变化等等。深入解析VLP体外组装机制，探索保持VLP结构稳定性和活性的策略，为VLP的高效分离纯化提供理论依据和指导。

类病毒颗粒(virus-like particles, VLPs)技术是现代新型疫苗的发展方向，在传染病和重大疾病的防治中具有重要应用前景。VLP通常含有多个亚基，亚基间由非共价键或易解离的二硫键连接，很容易在分离纯化过程中发生解聚或聚集，导致活性丧失，许多产品的活性收率不到20%甚至5%。本项目针对此问题，以乙型肝炎病毒表面抗原(HBsAg)为模型，研究溶液环境和固液界面对VLP结构的影响。利用动态光散射粒度仪等原位检测了不同溶液环境和层析介质条件下VLP组装结构的分子量、水力学半径和表面电荷的变化等等，筛选出了能够保持VLP活性的溶液条件，加速下游分离纯化程序，实现类病毒颗粒的结构稳定和活性保持。通过先进的分析技术如差示扫描量热和等温滴定量热技术研究了VLP结构变化的动力学和热力学参数，双偏振极化干涉仪原位检测了VLP在层析介质上的吸附动力学及构象变化，阐明了不同条件下HBsAg在固液界面上构象变化的规律及层析填料的性质对HBsAg组装体结构的影响规律。项目通过深入解析VLP体外组装机制，探索保持VLP结构稳定性和活性的策略，为VLP的高效分离纯化提供了理论依据和指导。