鼠李糖乳杆菌颗粒表面展示系统的构建

发布时间：2018-01-18 11:27

本文关键词：鼠李糖乳杆菌颗粒表面展示系统的构建　出处：《生物工程学报》2017年01期 　论文类型：期刊论文

【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,3.8×10~6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(P0.05)。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]:In order to compare the motif binding activity of different motifs, a new surface display system of Lactobacillus rhamnoides granules was constructed. Lactobacillus rhamnoides GEM(Gram-positive enhancer matrixGem granules were prepared by thermal acid treatment and observed by electron microscope. RT-PCR detection and SDS-PAGE detection were used to identify the treatment effect. At the same time, the anchored protein PA3-EGFP and P60-EGFP were expressed by E. coli and incubated with GEM particles. Finally, the binding efficiency of Lactobacillus rhamnoides GEM particles with anchoring protein was evaluated by Western blot, electron microscope, fluorescence microscope and fluorescence spectrophotometry. The inactivated peptidoglycan skeleton gem granules were obtained by treating Lactobacillus rhamnoides with 10% TCA. The copy number of DNA in 3.8 脳 10 ~ 6 GEM particles was only 32. The fusion proteins PA3-EGFP and P60-EGFP were anchored on GEM particles by Western blot and fluorescence microscopy. The results of fluorescence spectrophotometer showed that the binding efficiency of anchored protein PA3-EGFP to GEM was slightly higher than that of P60-EGFP. However, the difference was not significant (P 0.05). The above results showed that the binding efficiency of GEM granules made from Lactobacillus rhamnoides to the anchoring protein PA3P60 was good. It can be used to construct a new surface display system of exogenous protein and lay a foundation for the further research and application of bacterial-like granulocyte vaccine.
【作者单位】：天津农学院动物科学与动物医学学院;军事医学科学院生物工程研究所;
【分类号】：R392
【正文快照】： 鼠李糖乳杆菌(Lactobacillus rhamnosus GG,LGG)为革兰氏阳性菌,是研究最广泛的乳酸菌之一,具有生长速度快、生物安全性高等优点,能够有效调节肠道菌群和提高机体克疫力[1]。近些年,在基因工程技术的推动下,Qr于包括鼠李糖乳杆菌在内的多种乳酸菌(Lactis acid bacteria,LAB)已

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