【作者】 张莹莹；

【导师】 刘晨光；

【作者基本信息】 中国海洋大学 ， 生物工程（专业学位）， 2013， 硕士

【摘要】 S层蛋白是古菌和一些细菌细胞表面的一类蛋白质分子。S层蛋白有一种蛋白或糖蛋白形成晶格结构，罕见两种或以上的蛋白。S层蛋白与细胞外被通过非共价键结合，利用高浓度的氢键破坏试剂如脲素、氯化锂、盐酸胍等可将S层蛋白从细胞外被上解离下来，得到S层蛋白的亚单位。S层蛋白序列中非极性氨基酸含量比较高，赋予了S层蛋白的较高的疏水性，使得S层蛋白在除去变性剂后，能在溶液、气液界面、固体支持物或脂膜等界面上重新自组装成有序的晶格结构。乳酸菌是与人类关系密切的益生菌，能够维持肠道生态平衡，它的代谢产物能降低肠道pH,抑制病原菌在胃肠道的粘附、生长和产毒作用，具有防止便秘、帮助消化、增强机体免疫等功效。乳酸菌进入胃肠道，首先要定植在胃肠道上，才能发挥益生功能，粘附是首要的步骤。根据文献报道，乳酸菌的S层蛋白与其粘附过程有关。基于S层蛋白的自组装特性和乳酸菌S层蛋白的粘附性这两方面的认识，本论文旨在研究S层蛋白对药物输送载体的改良功效。即S层蛋白自组装在药物输送载体—海藻酸钙微球上，研究S层对微球的缓释及微球对肠道组织吸附性能的影响。实验中选择提取瑞士乳杆菌的S层蛋白，首先通过两种途径对S层蛋白的提取进行了优化：优化瑞士乳杆菌培养基，提高单位时间内菌体量；比较不同的变性剂在不同浓度、不同pH条件下的提取效果，使S层蛋白的纯度和量达到良好的水平。继而通过荧光显微镜、扫描电镜、SDS-PAGE检测S层蛋白对微球的吸附情况。在培养基优化实验中，首先通过Plackett-Burman实验设计筛选出了对菌体量具有显著影响的因素，分别是葡萄糖、牛肉膏和柠檬酸铵。然后进行了最陡爬坡实验逼近最佳响应区。最后利用Box-Behnken实验设计进行响应面优化后的最佳培养基组份为蛋白胨10g/L，酵母提取物5g/L，乙酸钠6.25g/L，吐温801.25g/L，磷酸氢二钾2.5g/L，牛肉膏8.96g/L，葡萄糖9.47g/L，柠檬酸铵3g/L，MgSO₄·7H₂O0.2g/L，MnSO₄·H₂O0.0625g/L。优化后的培养基使菌体干重提高了将近66%。不同变性剂脲素、盐酸胍、氯化锂在不同浓度和pH条件下S层蛋白提取效果的比较中，脲素提取的蛋白量很少，盐酸胍和氯化锂提取的蛋白量通过电泳检测差别不大，都比较多，但是盐酸胍提取方法杂蛋白较多，所以最后选择（pH=2）5M氯化锂提取S层蛋白。通过荧光显微镜、扫描电镜、SDS-PAGE方法证实了S层蛋白能吸附到海藻酸钙微球上，吸附过程发生的非常快，推测是引起吸附的作用力是静电引力。更多还原

【Abstract】 S-layer proteins represent the outmost component of many bacteria and archaea.Each S-layer protein is composed of a single protein or glycoprotein, scarcely of twoor more protein species, and forms crystalline arrays. The S-layer protein subunits arenon-covalently linked to each other as well as to the supporting cell wall, and can bedisintegrated into monomers by high concentration of hydrogen bond-disruptingagents such as urea，lithium chloride or guanidine hydrochloride. The content ofnon-polar amino acids of the S-layer sequences is relatively high, resulting in theirhydrophobic property. That is the reason of their ability of self-assemble,whichmeans isolated S-layer subunits can recrystallize into regular arrays in suspension,air-liquid interfaces, on solid supports, or lipid films and liposomes once thedisrupting agent used for their isolation has been removed.Lactic acid bacterial which can colonize human intestinal tract is beneficial tohuman body. They can improve or adjust intestinal microbial flora balance.Metabolites of them also have various benefits, such as reducing gut pH value,inhibiting spoilage bacterial growth and weakening their ability to produce poison,preventing constipations，helping digest, enhancing immunity. It has been reportedthat colonization of Lactic acid bacterial that is believed to be the first step forbenefits is related to surface layer proteins.On the basis of ability to self-assemble and be adhesive to intestinal tract, theobjective of the paper was to study whether the S-layer can improve properties ofdrug delivery carrier-alginate microspheres. That is to say when the S-layer assembledon the surface of alginate microspheres, the properties of sustained release andadhesion to tissues of microspheres were better or not. We chose Lactobacillushelveticus for isolation of S-layer proteins. First, we optimized the extraction methodsof the S-layer by two ways:（1）optimize the culture medium of Lactobacillushelveticus, for the purpose of increasing the amount of bacterial;（2）compare the extraction methods of different denaturing agents in different concentrations anddifferent pH values,in order to make purity and amount of S-layer proteins reach agood level. Then we studied the adsorption effect of S-layer proteins by fluorescencemicroscopy,scanning electron microscopy, and SDS-PAGE.In the experiment of optimization of culture medium, glucose, peptone, ammon-ium citrate tribasic were the three critical medium components among others by thePlackett-Burman design. The steepest ascent method was used to access the optimalregion of the medium composition. After using Box-Behnken design and response su-rface methodology, the optimal medium composition was achieved（g/L）: peptone,10;yeast extract,5;sodium acetate,6.25; Tween80,1.25; K2HPO₄,2.5; beef extract,8.96；glucose,9.47; ammonium citrate tribasic,3; MgSO₄·7H₂O,0.2; MnSO₄·H₂O,0.0625.In the experiment of comparisons among different extract methods, production ofS-layer proteins was the lowest by6M,8M,9M urea. Although the production ofprotein by2M,4M,6M guanidine hydrochloride and5M lithium chloride is aboutequal, the purity was better extracted by lithium chloride. So we chose （pH=2）5Mlithium chloride for protein extraction.Alginate microspheres absorption onto S-layer proteins was confirmed byfluorescence microscopy, scanning electron microscopy, and SDS-PAGE. The processtook place very quickly, so we presumed that the absorption process relied onelectrostatic force.更多还原