乳杆菌表层蛋白对脂质体的包被作用及互作机制研究

Lactobacillus surface layer proteins (SLPs) can be reassembled on liposomes to form a coating layer and improve the functional properties of liposomes. However, the effects of different structural SLPs on the functional properties of liposomes and their interaction mechanism with liposomes are not clear. In this project, glycosylated and non-glycosylated SLPs of Lactobacillus are supposed to be chosen as research objects. The structures and properties of SLPs are intended to be analyzed by proteomics methods. The best conditions for different structural SLPs to coat cationic liposomes will be screened. The effects of SLPs with different structures and properties on liposome stabilities will be studied, and the regularity will be analyzed. After liposomes are coated with SLPs, the structure changes of SLPs and liposomes will be investigated by fluorescence probe, infrared spectroscopy and nuclear magnetic resonance to reveal the interaction mechanism between SLPs and liposomes. Cellular and molecular biology techniques will be used to study the new biological functions that Lactobacillus SLPs confer on liposomes. In this project, the interaction mechanism between SLPs and liposomes will be revealed from a new perspective. And the improvement of liposomes properties by SLPs will be explored, which can provide a theoretical basis for the application of Lactobacillus SLPs in food and biomedical fields.

乳杆菌表层蛋白可在脂质体表面重新自组装形成包被层，改善脂质体的功能特性。但不同结构表层蛋白对脂质体功能特性影响的规律及其与脂质体的互作机制尚不明确。本项目拟选择糖基化和非糖基化乳杆菌表层蛋白作为研究对象，应用蛋白质组学手段对其进行结构和性质解析。筛选不同结构表层蛋白包被阳离子脂质体的最佳条件，考察表层蛋白结构性质差异对脂质体稳定性的影响，并分析其规律。采用荧光探针、红外光谱和核磁共振等方法，研究表层蛋白包被脂质体后双方结构特性的变化，揭示乳杆菌表层蛋白与脂质体的互作机制。通过细胞和分子生物学技术，研究乳杆菌表层蛋白赋予脂质体的新的生物学功能。本项目将从一个新的视角探索乳杆菌表层蛋白与脂质体的互作机制，发掘其对脂质体功能性质的改善，为乳杆菌表层蛋白在食品和生物医药领域的应用提供理论依据。

乳杆菌表层蛋白可在脂质体表面重新自组装形成包被层，改善脂质体的功能特性。但乳杆菌表层蛋白对脂质体功能特性影响的规律及其与脂质体的互作机制尚不明确。本项目选择Lactobacillus acidophilus ATCC 4356、L. buchneri BNCC 187961、 L. kefir BNCC 190565作为研究对象，采用5 M LiCl溶液提取表层蛋白并进一步通过离子交换色谱对提取物进行纯化，纯化后的表层蛋白结构与性质分析如下：3种乳杆菌表层蛋白的分子量都在43-46kDa，Periodic acid-schiff法检测发现L. buchneri BNCC 187961、L. kefir BNCC 190565的表层蛋白具有糖基；采用红外光谱测定表层蛋白的二级结构，发现3种表层蛋白α-螺旋与无规则卷曲含量相似，L. buchneri BNCC 187964表层蛋白有最多的β-折叠含量（46.5%）。差示扫描量热法测定表层蛋白的热变性温度分别为 L. acidophilus 67.50 ± 0.45°C； L. buchneri 78.34±0.67°C，L. kefir 70.21±0.91°C。氨基酸组成结果显示三种表层蛋白中碱性氨基酸的含量高于酸性氨基酸，符合乳杆菌表层蛋白的特性。通过测定Zeta电位和粒径确定3种表层蛋白包被阳离子脂质体的最佳条件为：L. kefir 100 μg/mL，25℃，80 min；L. acidophilus 30 μg/mL，25℃，160 min；L. buchneri 10 μg/mL，25℃，80 min。负染电镜下观察到3种表层蛋白在最适条件下，对脂质体都有完整的包被效果。通过构建包封荧光素的脂质体评价脂质体的稳定性，发现3种表层蛋白都可显著提高脂质体的热稳定性、pH稳定性、血清稳定性及储藏稳定性，且L. kefir表层蛋白的稳定性效果最好。红外光谱分析表明表层蛋白与脂质体之间是非共价相互作用，而且改变了表层蛋白的二级结构。此外，包被表层蛋白的脂质体还表现出对模拟胃肠道组织的高黏附性，以及对铁蛋白分子和IgG的结合作用。本项目构建了一个新的纳米载体系统，证实了乳杆菌表层蛋白对脂质体稳定性和功能性质的改善，为乳杆菌表层蛋白和脂质体在食品和生物医药领域的应用提供理论依据。