【作者】 王龙；

【导师】 王和平；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2005， 硕士

【摘要】 采用选择性培养基,从健康成年猪空、回肠段分离出 26 株菌株,经菌落形状、镜检及各种生化特征反应和糖发酵,确切鉴定出 5 株植物乳酸杆菌、4 株发酵乳酸杆菌、1 株嗜酸乳酸杆菌和 6 株大肠杆菌。分离出野生大肠杆菌的同时确定了空、回肠中的优势定植乳酸杆菌种类,为转β-甘露聚糖酶大肠杆菌在猪肠道中的高效表达以及该工程菌的改造和转基因微生态制剂的制备奠定了良好的基础。 随 后 将 质 粒 PT002-ManI 、 PUC18-Kil 和 PUC18-GFP ( Green Flourescent Protein)分别转化至野生大肠杆菌(YS)与工程菌(BL21) 形成 BL21-PT002-ManI/PUC18-Kil,YS- PT002-ManI/PUC18-Kil,BL21-PUC18-GFP 和 YS- PUC18-GFP。并通过绿色荧光蛋白指示特性以及目的基因检测技术,比较研究了野生大肠杆菌与工程菌间的生长与表达性能。 选择同一窝雄性猪只作十二指肠和回肠瘘管,将微胶囊化的转基因菌灌注液从十二指肠瘘管处注入,然后分别采集回肠消化样和新鲜粪便样进行菌落 PCR 和ELISA 双抗夹心法检测,结果表明灌注转基因菌不仅存活于整个消化道,而且能在回肠中表达出有活性的半纤维素酶。更多还原

【Abstract】 The 26 bacterial strains were isolated from jejunum and ileum of healthy and grown-up swine with some selective media .According to its shape of colony ,Gram stain and some biochemical characteristics as well as sugar fermentation reaction ,the preliminary identification was done, namely there are 5 strains of L.plantarum, 4 strains of L.fermentum, 1 strain of L.acidophilus and 6 strains of E.coli.The kind of dominant colonic Lactobacillus in jejunum and ileum was determined.So the researth provided a good foundations for expressing of transgenic E.coli(?-mannase) in swine effectively and reforming of the engineering bacteria and making probiotics and microecologics. The BL21-PT002-ManI/PUC18-Kil,YS- PT002-ManI/PUC18-Kil,BL21-PUC18-GFP and YS- PUC18-GFP have been established by transforming plasmids PT002-ManI,PUC18-Kil and PUC18-GFP(Green Flourescent Protein)to YS and BL21. The bacteria relatively accustomed to expression in vitro has been selected by researching characteristics of growth and expression on YS and BL21 with GFP characters and gene examination techniques. After swines have been selected to make cannulas at duodenum and ileum,the bacteria have been made microcapsule and then blent with others to become the pouring liquids to pour into swines through the cannula. The specimens have been examinated with colony PCR and ELISA .更多还原