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

本研究旨在对山羊化脓隐秘杆菌重庆分离株的胶原结合蛋白(collagen-binding protein,Cbp)CbpA(CbpACQ)进行序列分析,鉴定其中2个潜在的肝素结合结构域NRB和B1。运用生物信息学软件分析CbpA-CQ基因及其编码产物。采用PCR扩增NRB和B1基因,将其克隆至原核表达载体pGEX-4T-1。融合蛋白GST-NRB和GST-B1在大肠杆菌DE3菌株中被诱导表达,使用Gluthathione-Sepharose 4B纯化。采用免疫印迹检测融合蛋白GST-NRB、GST-B1与HeLa细胞的黏附情况,以及肝素对融合蛋白黏附细胞的抑制情况。结果显示,重庆株CbpA有7个胶原结合蛋白B结构域,约占全长的55%,此结构域与化脓隐秘杆菌、链球菌属等细菌有同源性。重庆株CbpA与化脓隐秘杆菌的CbpA在进化树中聚集成一个进化支。从诱导菌裂解物上清中亲和纯化得到融合蛋白GST-NRB和GST-B1。GST-NRB、GST-B1呈剂量依赖性黏附HeLa细胞,肝素呈剂量依赖性抑制GST-NRB、GST-B1黏附HeLa细胞。结果表明尽管CbpA-CQ有很大变异性,但具有已知化脓隐秘杆菌CbpA的共同序列特征;其NRB和B1区域为肝素结合结构域。

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 CbpA-CQ 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.