鼠李糖乳杆菌颗粒表面展示系统的构建

Construction of Lactobacillus rhamnosus GG particles surface display system

摘要:

为了比较不同锚钩蛋白基序结合活性，构建新型的鼠李糖乳杆菌颗粒表面展示系统。首先，用热酸处理法制备鼠李糖乳杆菌GEM (Gram-positive enhancer matrix，GEM) 颗粒，并通过电镜观察、RT-PCR检测和SDS-PAGE检测鉴定其处理效果；同时，利用大肠杆菌表达了锚定蛋白PA3-EGFP和P60-EGFP并将其与GEM颗粒共同孵育结合；最后，使用免疫印迹、电镜观察、荧光显微镜观察和荧光分光光度法评价鼠李糖乳杆菌GEM颗粒与锚定蛋白的结合效率。结果表明，使用10%的TCA处理鼠李糖乳杆菌得到了灭活的肽聚糖骨架 (GEM颗粒)，经鉴定其大小形态均一，绝大部分无蛋白残留，3.8×106个GEM颗粒样品中的DNA拷贝数仅为32；免疫印迹和荧光显微镜观察均可检测到融合蛋白PA3-EGFP和P60-EGFP锚定在GEM颗粒上，且结合在GEM颗粒表面的锚定蛋白呈絮状。荧光分光光度计法检测结果显示锚定蛋白PA3-EGFP结合GEM的效率稍高于P60-EGFP，但差异不显著 (P>0.05)。以上结果表明由鼠李糖乳杆菌制得的GEM颗粒与锚定蛋白PA3、P60的结合效率良好，可用于构建新型的外源蛋白表面展示系统，进而为后续的细菌样颗粒疫苗的研究与应用奠定基础。

Abstract:

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.