枯草芽胞杆菌DarA蛋白的鉴定及其原核表达

doi:10.11843/j.issn.0366-6964.2021.05.023

摘要/Abstract

摘要： 目前，我国全面禁止饲料中添加抗生素，寻找新型的抗生素替代物是当前研究的热点之一。本研究旨在分离枯草芽胞杆菌SXAU18所产具有抗菌活性的蛋白物质，并通过原核表达系统获得其具有抗菌活性的重组蛋白。试验选用硫酸铵沉淀、氯仿抽提、分子截留等蛋白提取技术，对枯草芽胞杆菌SXAU18产生的抗菌蛋白进行分离纯化；通过SDS-PAGE技术和牛津杯扩散法检测分离到的蛋白物质的抑菌活性；通过质谱、生物信息学技术分析预测目的蛋白的基因序列；利用大肠杆菌表达系统和AKTA蛋白纯化系统对目的蛋白进行表达和纯化；借助牛津杯扩散法鉴定重组蛋白的抑菌活性。结果表明，枯草芽胞杆菌SXAU18所产的抗菌物质为相对分子质量15 ku左右的蛋白质，含有共同的特定氨基酸序列—GSSIFGLAPGK，对金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特菌有较好的抑菌活性；通过质谱分析和生物信息学推测SXAU18所产的（主要）抑菌蛋白为DarA；经诱导表达的重组蛋白DarA主要以可溶性上清蛋白形式存在，纯化后的DarA蛋白为单一条带，并具有良好的抑菌活性。结果提示，分离自枯草芽胞杆菌SXAU18的抗菌蛋白DarA具有抑制金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特菌生长的活性，且可以通过体外表达获取该蛋白。

关键词: 羊驼, 枯草芽胞杆菌, 细菌素, 分离纯化, 原核表达

Abstract: At present, the addition of antibiotics in animal feed is completely banned in China, thus particular attention has been focused on finding novel antibiotic substitutes in the field. Hence, we aimed to isolate the bacteriocin with antibacterial activity produced by Bacillus subtilis SXAU18, and obtain its antibacterial recombinant protein using prokaryotic expression system. In this study, we isolated the target protein by (NH₄)₂SO₄precipitation, chloroform extraction, ultrafiltration and tested its antibacterial activity by SDS-PAGE analysis and Oxford cup diffusion method. Predicted protein was expressed and purified by prokaryotic expression system and AKTA system. The antibacterial activity of the recombinant protein was determined by the Oxford cup diffusion method. We identified a 15 ku protein containing a unique peptide sequence GSSIFGLAPGK from the antibacterial substance produced by Bacillus subtilis SXAU18, which exerts pronounced antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes. The mass spectrometry and bioinformatics analysis indicated that the 15 ku protein is DarA. Recombinant expression of DarA in prokaryotic expression system and its purification revealed that the DarA was mainly expressed in a soluble form and could be purified to homogeneity, which showed as a single band. The antibacterial test showed that the recombinant DarA has antibacterial activity. The results indicate that DarA from Bacillus subtilis SXAU18 have antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes, and this protein can be obtained through recombinant expression.

Key words: alpaca, Bacillus subtilis, bacteriocin, isolation and purification, prokaryotic expression

中图分类号:

Q939.92

于秀菊, 孙铮, 韩小涛, 李钰钰, 于淼, 董常生. 枯草芽胞杆菌DarA蛋白的鉴定及其原核表达[J]. 畜牧兽医学报, 2021, 52(5): 1378-1385.

YU Xiuju, SUN Zheng, HAN Xiaotao, LI Yuyu, YU Miao, DONG Changsheng. Identification of DarA Protein of Bacillus subtilis and Its Recombinant Expression Using Prokaryotic Expression System[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1378-1385.

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