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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,3.8×10~6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。
In order to compare the binding activity of different anchoring protein motifs, a novel Lactobacillus rhamnosus particle surface display system was constructed. First, gelatin-positive enhancer matrix (GEM) particles were prepared by hot-acid treatment. The effect of treatment was evaluated by electron microscopy, RT-PCR and SDS-PAGE. The anchoring proteins PA3-EGFP and P60-EGFP were co-incubated with GEM particles. Finally, immunoblot, electron microscopy, fluorescence microscopy and fluorescence spectroscopy were used to evaluate the relationship between Lactobacillus rhamnosus GEM particles and anchored protein The binding efficiency. The results showed that in vitro treatment of Lactobacillus rhamnosus with 10% TCA gave inactivated peptidoglycan backbone (GEM particles), which were identified as being of uniform size and shape with no protein residue. 3.8 × 10 ~ 6 GEM particles The DNA copy number in the sample was only 32; both the immunoprecipitation and fluorescence microscopy showed that the fusion proteins PA3-EGFP and P60-EGFP were anchored on the GEM particles and the anchored proteins bound to the GEM particles were flocculent. The results of fluorescence spectrophotometer showed that the efficiency of anchoring protein PA3-EGFP combined with GEM was slightly higher than that of P60-EGFP, but the difference was not significant (P> 0.05). The above results show that GEM particles prepared from Lactobacillus rhamnosus have a good binding efficiency with the anchoring proteins PA3 and P60 and can be used to construct a novel exogenous protein surface display system for the subsequent research and application of bacterial particle vaccines Lay the foundation.