摘要：芽孢杆菌是一种革兰氏阳性菌，它能够分泌大量的蛋白到细胞外，而且在此过程中其细胞壁不会产生内毒素。多数芽孢杆菌对人畜无致病性，而且，其细胞壁成分主要是肽聚糖和磷壁酸，在分泌蛋白的过程中不会产生诸如多糖类等难以除去的物质。因此，芽孢杆菌表达系统是一种重要的外源基因表达系统。本课题的研究中，通过收集全国十余省市的土壤、牛奶样品，鉴定分离地衣芽孢杆菌和枯草芽孢杆菌共4株。希望能够以此菌株作为载体，构建高效表达外源基因的表达系统。此外，为了提高在枯草芽孢杆菌中胞外蛋白酶的表达效率，本课题以枯草芽孢杆菌168为基础，采用无痕敲除的方法，构建了一株8个主要胞外蛋白酶基因nprE、aprE、epr、bpr、mpr、nprB、vpr、wprA启动子区域和部分基因敲除的菌株。30387
毕业论文关键词：芽孢杆菌；表达系统；无痕敲除；筛选
The screening and modification of Bacillus expression system
Abstract：Bacillus is a gram-positive bacterium, it can secrete large amounts of protein to the outside of the cell, and its cell wall in the process won't produce endotoxin Most bacillus no pathogenicity for human, and its cell wall components mainly, peptidoglycan and teichoic acid in the process of secretory protein does not produce such as polysaccharide, difficult to remove material Therefore, bacillus expression system is a kind of important heterologous gene expression system in the study of this topic, soil milk samples collected from more than 10 provinces and cities nationwide, 6 strains of bacillus licheniformis appraisal separation Hope to be able to this strain as the carrier, construct efficient expression of exogenous gene expression system In addition, in order to improve the extracellular protease in bacillus subtilis expression efficiency, this topic is based on bacillus subtilis 168, adopt the method of non-trace knockout, builds a plant eight major extracellular protease gene nprE、aprE、epr、bpr、mpr、nprB、vpr、wprA strains of defects.
Key words: Bacillus; expression system; non-trace knock out.
目  录
摘要.1
关键词.1
Abstract.1
Key words.1
引言    3
1  材料与方法    4
1.1  材料及其培养    4
1.1.1 液体培养    4
1.1.2 固体培养    5
1.1.3 脱脂牛奶培养基    5
1.1.4 菌种保藏    5
1.2  地衣芽孢杆菌的分离筛选    5
1.2.1 土壤样品采集    5
1.2.2 土壤样品处理    5
1.2.3 土壤微生物分离纯化    5
1.2.4 土壤微生物鉴定    5
1.3  胞外蛋白酶基因的敲除    6
1.3.1 引物设计    6
1.3.2 同源臂的获得    7
1.3.3 同源臂的融合    7
1.3.4 融合片段转化    8
1.3.5 感受态的制作    9
1.3.5.1 感受态细胞的培养与转化    9
1.3.5.2 培养基配方    9
1.4 生长曲线的测定    9
1.5 无痕敲除方法技术路线    9
2  结果与分析    11
2.1  土壤样品分离菌株鉴定    11
2.2  nprE基因敲除鉴定    13
2.3  aprE基因敲除鉴定    14
2.4  epr基因敲除鉴定    15
2.5  bpr基因敲除鉴定    16
2.6  mpr基因敲除鉴定    17
2.7  nprB基因敲除鉴定    18
2.8  vpr基因敲除鉴定    19
2.9  wprA基因敲除鉴定    20 芽孢杆菌表达系统的筛选和改造:http://www.751com.cn/shengwu/lunwen_26065.html