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

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (1): 173-180.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

沈克飞¹, 许国洋¹, 胡瑞思², 徐登峰¹, 杨睿¹, 付利芝¹, 张素辉^1*

1. 重庆市畜牧科学院, 重庆 402460;
2. 西北农林科技大学动物医学院, 杨凌 712100

收稿日期:2017-06-30 出版日期:2018-01-23 发布日期:2018-01-23
通讯作者: 张素辉,副研究员,主要从事病原学与分子生物学研究,E-mail:446116409@qq.com
作者简介:沈克飞(1980-),男,安徽六安人,硕士,主要从事分子致病机理研究,E-mail:coffeeshine@163.com
基金资助:
重庆市基础与前沿研究计划项目（cstc2015jcyjA80035）；国家自然科学基金委员会青年基金项目（31402149）；重庆市基本科研业务费项目（16417）

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

SHEN Ke-fei¹, XU Guo-yang¹, HU Rui-si², XU Deng-feng¹, YANG Rui¹, FU Li-zhi¹, ZHANG Su-hui^1*

1. Chongqing Academy of Animal Sciences, Chongqing 402460, China;
2. College of Veterinary Medicine, Northwest A & F University, Yangling 712100, China

Received:2017-06-30 Online:2018-01-23 Published:2018-01-23

摘要/Abstract

摘要：

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（CbpA-CQ）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。

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.

中图分类号:

S852.61

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.

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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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

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