ST融合蛋白的酵母表面展示及其对大鼠小肠黏膜结构的影响  

Yeast surface display of ST fusion protein and its effect on intestinal mucosal structure in rats

作　　者：沙洲[1] 李诗语[2] 卢晓冉 王莉[1] 卜昭阳[2,3] 王兴龙 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医学报》2018年第2期316-325,共10页Chinese Journal of Veterinary Science

摘　　要：本试验旨在获得能够将大肠杆菌ST融合蛋白展示在酿酒酵母表面的重组酿酒酵母基因工程菌并探究其对大鼠小肠黏膜结构的影响。将estA和estB基因克隆至pYD1质粒中，构建重组质粒pYD1-e5tA-estB，应用酵母表面展示技术获得EBY-100／pYD1-estA-estB重组酵母基因工程菌。选取36只SPF级SD大鼠，随机分为空白对照组、工程菌组、酵母菌组，分别灌胃生理盐水、工程菌菌液、酿酒酵母菌菌液，持续灌胃21d后连续3d灌胃大肠杆菌H10407菌液，取各组大鼠的十二指肠、空肠、回肠制作切片，观察各段小肠绒毛的长度、宽度、隐窝深度及绒毛长度／隐窝深度（V／c）进行统计分析。利用EKISA、real—timePCR技术观察肠道黏膜中的SIgA、IL2、IL-4和IFN7含量变化。结果显示，通过免疫荧光试验证明大肠杆菌ST融合蛋白成功在酿酒酵母表面展示；灌胃21d时，与空白对照组相比，工程菌组及酵母菌组的十二指肠绒毛长度、v／c值显著升高（P〈0．01），工程菌组绒毛宽度显著升高（P〈0．05）；空肠绒毛长度、v／c值显著升高（P〈0．01），隐窝深度显著下降（P〈0．05）；工程菌组和酵母菌组的回肠绒毛长度及V／c值显著升高（P〈0．01）。灌胃大肠杆菌后，各组与灌胃前相比，空白对照组及酵母菌组的十二指肠与空肠绒毛长度、V／c值显著下降（P〈0．01），空肠隐窝深度显著升高（P〈0．01），回肠V／c值显著下降（P〈0．01）；工程菌组无明显变化。灌胃21d，工程菌组与酵母菌组的SIgA、IL-2、IL-4和IFN-7含量均显著高于空白对照组（P〈0．01）；攻毒大肠杆菌后，空白对照组及酵母菌组的SIgA、IL-2和IL-4含量均显著下降（P〈0．01），各组的IFN-γ含量显著升高（P〈0．01）。结果表明，EBY-100／pYD1-estA-estB重组酵母基因．72程菌不仅具有酵母菌的益生作用，能促进小肠黏膜发育，而且对�The aim of the study is to obtain recombinant gene-engineering Saccharomyces cerevisiae which can demonstrate enterotoxigenic Escherichia coli ST fusion protein on its surface and to in- vestigate the effect of recombinant gene-engineering Saccharomyces cerevisiae on intestinal muco- sal structure in rats. With cloning estA and estB genes into pYD1 plasmid to construct pYDl-estA- estB plasmid, the S. cerevisiae surface display system of EBY-lOO/pYDl-estA-estB was successful- ly constructed by the yeast surface display technology. SPF SD rats of 36 were divided randomly into 3 groups：control, gene-engineering S. cerevisiae and S. cerevisiae groups. Each of rats from the three groups was respectively gavaged saline,gene-engineering S. cerevisiae culture and S. cer- evisiae culture. After 21 days, the three groups were gavaged enterotoxigenic Escherichia coli H10407 for 3 days, slice of duodenum,jejunum, ileum were made by paraffin and recorded the length, width,crypt depth and villus length/crypt depth（V/C） for statistical analysis. The levels of SIgA,IL-2, IL-4 and IFN-γ in the intestinal mucosa were measured by ELISA and real-time PCR. The fusion protein was successfully expressed on the surface of S. cerevisiae by the immune fluo- rescence. After 21 days,compared with the control group,the duodenal villus length and V/C of gene-engineering S. cerevisiae and S. cerevisiae groups were extremely significantly inereased（P〈 0.01）, villous with of gene-engineering S. cerevisiae group were significantly increased（P%0.05）, jejunum villus length and V/C were extremely significantly higher（P〈0.01） ,crypt depth signifi- cantly decreased（P〈0.05） ,villus length and V/C of gene-engineering S. cerevisiae and S. cerevi- siae groups were extremely significantly increased（P〈0.01）. Compared with after and before ga- raging,the stomach,duodenum and jejunum villus length, V/C, the control group and the S. cere- visiae group were extremely significantly decrease（P〈0.01）, crypt depth of jejunum

关 键 词：ST 产肠毒素大肠杆菌 酿酒酵母 表面展示 大鼠 小肠黏膜 

分 类 号：S853.61[农业科学—临床兽医学] R535[农业科学—兽医学]