化脓隐秘杆菌肝素结合蛋白的提取及铁结合蛋白的黏附特性分析

doi:10.11843/j.issn.0366-6964.2022.01.024

摘要/Abstract

摘要： 肝素可剂量依赖性地抑制化脓隐秘杆菌（Trueperella pyogenes）黏附宿主细胞。本研究旨在认识化脓隐秘杆菌肝素结合蛋白及其黏附特性，采用肝素琼脂糖凝胶从化脓隐秘杆菌裂解物中提取蛋白，运用蛋白质质谱和免疫印迹对所提取的蛋白质进行鉴定。采用免疫印迹检测所提取蛋白质与自然感染化脓隐秘杆菌山羊血清的反应原性。制备重组蛋白，对重组蛋白的的肝素结合活性及肝素、抗血清对肝素结合活性的抑制作用进行了分析，对重组蛋白细胞黏附活性和抗血清抑制化脓隐秘杆菌黏附宿主细胞活性进行了分析。结果表明，提取到包含铁结合蛋白（iron-binding protein，IBP）在内的多种ATP结合盒（ATP binding cassette）转运体的底物结合蛋白（substrate binding proteins）。在自然感染山羊血清中检测到抗IBP的IgG抗体，表明IBP在化脓隐秘杆菌感染过程中表达。SDS-PAGE结果显示，rIBP能结合肝素琼脂糖凝胶，生理浓度的NaCl能部分解离与肝素琼脂糖凝胶结合的rIBP。免疫印迹结果显示肝素能剂量依赖性地抑制rIBP结合肝素琼脂糖凝胶，表明rIBP特异性结合肝素。rIBP能黏附MDBK细胞。免疫印迹显示rIBP抗血清不能明显抑制rIBP结合肝素，但活菌计数结果显示能抑制化脓隐秘杆菌黏附MDBK细胞。本研究为深入认识化脓隐秘杆菌肝素结合蛋白及其在化脓隐秘杆菌黏附宿主细胞过程中的作用奠定了基础。

关键词: 化脓隐秘杆菌, 肝素结合蛋白, 铁结合蛋白, 黏附, 肝素

Abstract: Heparin could inhibited the adhesion of Trueperella pyogenes to host cells in a dose-dependent manner. To understand the heparin binding proteins from T. pyogenes and their adhesion characteristics, the proteins were extracted from the lysate of T. pyogenes by heparin aga-rose gel, and were identified by protein mass spectrometry and Western blotting. The reactivity of the extracted proteins with the serum from a goat naturally infected with T. pyogenes was detected by Western blotting. The recombinant proteins were prepared. The heparin binding activity of recombinant proteins and the inhibitory effect of heparin or the antiserum on the binding of recombinant proteins to heparin were analyzed. The cell adhesion activity of the recombinant proteins and the inhibitory effect of the antiserum on the adhesion of T. pyogenes to host cells were analyzed. The results showed that multiple substrate binding proteins of ATP binding cassette transporters including iron-binding protein (IBP) were extracted. The IgG antibody against IBP was detected in the serum of naturally infected goat with T. pyogenes, which indicated that IBP was expressed in the process of T. pyogenes infection. The result of SDS-PAGE showed that rIBP could bind heparin agarose gel, and the physiological concentration of NaCl could partially dissociate the rIBP bound to heparin agarose gel. The result of Western blotting showed that heparin could inhibit rIBP binding heparin agarose gel in a dose-dependent manner, indicating that rIBP specifically binds heparin. Western blot showed that rIBP could adhere to MDBK cells. Western blotting results showed that anti-rIBP serum could not inhibit the binding of rIBP to heparin, but the results of viable bacteria counting showed that anti-rIBP serum could inhibit the adhesion of T. pyogenes to MDBK cells. The result of this study laid a foundation for further understanding heparin binding proteins of T. pyogenes and their role in the adhesion of T. pyogenes to host cells.

Key words: Trueperella pyogenes, heparin binding proteins, iron-binding protein, adhesion, heparin

中图分类号:

S852.61

沈克飞, 许国洋, 付利芝, 杨柳, 牟豪, 张素辉. 化脓隐秘杆菌肝素结合蛋白的提取及铁结合蛋白的黏附特性分析[J]. 畜牧兽医学报, 2022, 53(1): 241-249.

SHEN Kefei, XU Guoyang, FU Lizhi, YANG Liu, MOU Hao, ZHANG Suhui. Extraction of Heparin Binding Proteins from Trueperella pyogenes and Adhesion Characteristics of Iron Binding Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 241-249.

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