猪链球菌细胞壁蛋白SSU05_1000的表达、纯化及活性分析  

Expression,purification and activity assay of Streptococcus suis cell wall protein SSU05_1000

作　　者：王涛[1,2] 郑玉玲[2] 袁媛[2] 江华[2] 郝淮杰[3] 李雪琴[2] 姜永强[1,2] 

机构地区：[1]安徽医科大学研究生学院,合肥230032 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [3]中国科学院微生物研究所病原微生物与免疫学重点实验室,北京100101

出　　处：《中国人兽共患病学报》2013年第8期757-761,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(No.81171528)资助~~

摘　　要：目的构建猪链球菌细胞壁蛋白SSU05_1000基因的重组表达质粒,在大肠杆菌BL21(DE3)中高效表达,并研究其对细胞间紧密连接的调控作用。方法以05ZYH33基因组为模板设计引物,构建pET30a-SSU05_1000重组表达质粒,转化到大肠杆菌中诱导表达,用镍亲和层析的方法对目的蛋白进行纯化,Western blot检测其对人脑微血管内皮细胞紧密连接的调控。结果构建的SSU05_1000重组表达质粒能够在大肠杆菌中高效表达,纯化的SSU05_1000蛋白纯度达90%以上,Western blot结果显示其与人脑微血管内皮细胞(hCMEC/D3)相互作用后,可以下调细胞间紧密连接蛋白Claudin-5的表达。结论 SSU05_1000蛋白能够破坏人脑微血管内皮细胞的紧密连接结构,提示其在猪链球菌穿血脑屏障(BBB,bloodbrain barrier)过程中起重要作用。The role of recombinant SSU05_1000 protein on the regulation of tight junctions is investigated in this study. The SSU05_1000 gene was amplified using the primers designed according to 05ZYH33 genome sequences and cloned to the ex- pression vector pET30a to construct recombinant plasmid. The plasmid was transformed into E. coli BL21 （DE3） and induced to express by IPTG. The recombinant protein was purified by nickel affinity chromatography. Results showed that the recom binant expression plasmid could be highly expressed in E. coli and the purity of recombinant SSU05_1000 protein was above 90%. The purified protein was then treated withTriton X-114 to remove the contamination of endotoxin. Then, the human brain microvascular endothelial cells （hCMEC/D3） were treated by SSU05_1000 for different time and tested the expression of tight junctions by Western blot. The Western blot results show that SSU05_1000 could down-regulate the expression of tight junction protein Claudin-5 in hCMEC/D3, which indicates that SSU05_1000 protein might play important role on the traversal of S. suis across the human blood-brain barrier.

关 键 词：猪链球菌 SSU05_1000 紧密连接 

分 类 号：R378[医药卫生—病原生物学]