饲料植物乳酸杆菌食品级酸诱导表达系统的构建

To construct a acid inducible food-grade expression system in lactobacillus plantarum for feed，first of all，lactobacillus plantarum will be isolated from fermented feed or Silage or microbial ecological agent， and will be identified on the basis of morphology, physiological and biochemical feateues with 16S rDNA sequence analysis. Select a hopeful host strain for food-grade gene engineering by electrotransform、cryptic plasmid free、nisin sensitive experiments. Then, the functional replication DNA sequence of plasmid from lactobacillus plantarum will be identified by complete sequence analysis and deletion-insertion experiments。The replicon and promoter of vector pLEB590 will have been exchanged by above replicon and P170，as a construct food-gradc expressing system with nisin immunity selection marker．.Lactobacillus are the better choice of host strain for food-grade gene engineering due to their important activity in fermented feed、Silage and microbial ecological agent. In view of the big commercial profits of the species Lactobacillus plantarum which have been widely used in fermented feed、Silage and microbial ecological agent, construct a acid inducible food-grade expression system in lactobacillus plantarum，will promote the use 0f animal feed.

为了构建食品级酸诱导植物乳酸杆菌表达系统，先从发酵饲料、青贮、微生态制剂中分离植物乳酸杆菌，经生理、生化及16SrDNA鉴定后，通过自身质粒测定、电转化试验及nisin敏感试验筛选基因工程受体菌株；然后，经过可用于表达载体的植物乳酸杆菌质粒的分离和筛选、质粒DNA的测序和生物信息学分析、缺失和插入实验方法确定复制子；最后，用上述复制子代替乳酸球菌表达质粒pLEB590中的pSH71复制子，用酸诱导启动子P170更换上重组质粒的P45启动子，构建具有nisin抗性筛选标记的食品级酸诱导植物乳酸杆菌表达系统。.植物乳酸杆菌在发酵饲料、青贮饲料及微生态制剂中发挥着重要作用。构建食品级酸诱导植物乳酸杆菌表达系统，使该菌有自身及其各种代谢产物对机体有营养和免疫功能外,还具备其上携带的目的蛋白的专一性作用,成为集多种功能于一体基因工程菌。这对改善饲料质量，提高饲料的消化利用率具有重要意义。