胸膜肺炎放线杆菌三聚体自转运黏附素功能区的表达及黏附活性  

Expression and adhesive activity of functional domain of trimeric autotransporter adhesins in A.pleuropneumoniae

作　　者：周靓[1] 雷连成[1] 谢芳[1] 李树清[2] 李林溪[1] 韩文瑜[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]上海出入境检验检疫局,上海200135

出　　处：《中国兽医学报》2011年第3期342-346,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30870089;31072133)

摘　　要：试验对三聚体自转运黏附素(TAAs)的重要功能区第1 703～2 018氨基酸残基区域进行原核表达,并利用激光共聚焦显微镜观察重组蛋白在猪肺上皮细胞的结合部位,通过黏附抑制试验研究该蛋白的生物活性。结果显示,扩增的adh4基因片段与GenBank中相应的基因序列的同源性达100%,表达得到相对分子质量约为31 000的Adh4蛋白,该蛋白能够黏附在猪肺上皮细胞膜表面。猪肺上皮细胞经Adh4蛋白处理后,降低了APP对它的黏附能力,同时Adh4抗体可以抑制该菌对细胞的黏附,黏附菌数与其稀释度呈负相关。结果表明,Adh4蛋白可以介导APP对猪肺上皮细胞的黏附作用,进一步证明了位于N端1703～2018氨基酸残基区域是三聚体自转运黏附素蛋白的功能区,为研究胸膜肺炎放线杆菌的黏附机制奠定了基础。Adherence is an important process in bacterial pathogenicity.In order to make deep study on APP adherence to swine lung epithelial cells line mediated by trimeric atuotransporter adhesion（TAAs）,the important functional domain（amino acids 1703-2018） of trimeric atuotransporter adhesin from A.pleuropneumoniae serotypes 5 strains was expressed by E.coli strain BL21（DE3）.Then the interaction of recombinant protein with swine lung epithelial cell were examined under a confocal laser scanning microscope,and its biological activity was tested by adhesive inhibition assay.The results showed that the sequence identities were 100%between amplified adh4 gene and that from GenBank（CP000569.1）,and the size of recombinant Adh4 protein was about 31 000.Also,Adh4 protein could attach to the surface of bacteria,lung epithelial cell pretreated with Adh4 protein resulted in partial inhibition of APP adherence,and APP pretreated with antisera against Adh4 reduced adherence to epithelial cell,which seem negatively correlated with its dilution-fold.These observations demonstrate that Adh4 mediated APP adherence to swine lung epithelial cells line,indiating that the predicted fragment（amino acids 1 703-2 018） of TAAs is the functional domain.The data will further contribute to study for APP adhesion mechanism.

关 键 词：胸膜肺炎放线杆菌 三聚体自转运黏附素 重组蛋白Adh4 黏附 

分 类 号：S852.61[农业科学—基础兽医学]