山羊化脓隐秘杆菌重庆分离株CbpA肝素结合结构域鉴定

doi:10.11843/j.issn.0366-6964.2018.01.020

Abstract

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 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.

CLC Number:

S852.61

SHEN Ke-fei, XU Guo-yang, HU Rui-si, XU Deng-feng, YANG Rui, FU Li-zhi, ZHANG Su-hui. Identification of Heparin Binding Domains in CbpA of Trueperella pyogenes Chongqing Isolate from Goat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 173-180.

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Identification of Heparin Binding Domains in CbpA of Trueperella pyogenes Chongqing Isolate from Goat

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