畜牧兽医学报2018，Vol.49Issue(1)：173-180,8.DOI:10.11843/j.issn.0366-6964.2018.01.020
山羊化脓隐秘杆菌重庆分离株CbpA肝素结合结构域鉴定
Identification of Heparin Binding Domains in CbpA of Trueperella pyogenes Chongqing Isolate from Goat
摘要
Abstract
This study was aimed to analyze the sequences of collagen-binding protein A (CbpA) of Trueperella pyogenes Chongqing isolate from goat (CbpA-CQ) and identify the two potential heparin binding domains of NRB and B1.The sequences of CbpA-CQ gene and its coding product were analyzed by bioinformatics software.NRB and B1 genes were amplified by PCR and cloned into prokaryotic expression vector (pGEX-4T-1).The fusion proteins (GST-NRB and GST-B1) were induced in Escherichia coli strain DE3 and purified using Gluthathione-Sepharose 4 B beads.The adhesion of fusion proteins (GST-NRB and GST-B1) to HeLa cells and the adhesion inhibition of heparin were detected by Western blot.The results showed that the CbpA-CQ had 7 collagen-binding B domains covering 55 ％ in the whole sequence of CbpA,and the domains had homology with those of bacteria,such as T.pyogenes,Staphylococcus.CbpA-CQ and those of other T.pyogenes strains were clustered into a clade in the phylogenetic tree.Fusion proteins (GST-NRB and GST-B1) were purified by affinity purification from supernatant of induced bacteria lysate.The 2 proteins adhered to HeLa cells in a dose dependent manner,but the adhesion was inhibited by heparin in a dose dependent manner.The results suggest that although CbpACQ has large variability,it has common sequence characteristics compared with the published CbpA sequences.Besides,the NRB and B1 regions of CbpA-CQ are heparin binding domains.关键词
化脓隐秘杆菌/CbpA/黏附/肝素/肝素结合结构域Key words
Trueperella pyogenes/CbpA/adhesion/heparin/heparin binding domain分类
农业科技引用本文复制引用
沈克飞,许国洋,胡瑞思,徐登峰,杨睿,付利芝,张素辉..山羊化脓隐秘杆菌重庆分离株CbpA肝素结合结构域鉴定[J].畜牧兽医学报,2018,49(1):173-180,8.基金项目
重庆市基础与前沿研究计划项目(cstc2015jcyjA80035) （cstc2015jcyjA80035）
国家自然科学基金委员会青年基金项目(31402149) （31402149）
重庆市基本科研业务费项目(16417) （16417）
