枯草芽孢杆菌细菌素的分离、表达及稳定性分析

doi:10.11843/j.issn.0366-6964.2024.01.030

摘要/Abstract

摘要： 旨在分离枯草芽孢杆菌及其所产细菌素，并分析重组表达后的细菌素的抑菌活性和稳定性。通过牛津杯扩散法筛选羊驼粪便中高产细菌素的芽孢杆菌；借助16S rRNA鉴定分离菌；采用硫酸铵沉淀、氯仿抽提、SDS-PAGE、质谱分析等技术获得细菌素的氨基酸序列；利用大肠杆菌表达系统对细菌素进行体外表达，并利用牛津杯扩散法测定其抑菌活性和稳定性。结果显示，分离菌是一种枯草芽孢杆菌，命名为枯草芽孢杆菌SXAU18，其可产生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细菌素；枯草芽孢杆菌SXAU18所产细菌素可能是分子量为10~20 ku的DarA蛋白和两种未知蛋白，属于类细菌素的范畴，将未知蛋白分别命名为BLIS SXAU181和182；经原核表达的BLIS SXAU181和182主要以可溶性上清蛋白形式表达，纯化后为单一条带；重组BLIS SXAU181蛋白没有抑菌活性，重组BLIS SXAU182蛋白具有良好的抑菌活性，且具有耐高温、耐酸碱、耐人工胃液和肠液的的特性。综上，本研究从枯草芽孢杆菌SXAU18分离到具有抑制革兰阳性菌生长活性的细菌素，且重组表达后的BLIS SXAU182具有良好的抑菌活性和和稳定性。

关键词: 枯草芽孢杆菌, 细菌素, 分离, 表达, 稳定性

Abstract: The aim of the present study was to isolate Bacillus subtilis and its bacteriocins, and to analyse the antibacterial activity and stability of the recombinant bacteriocin. The Bacillus spp. producing bacteriocins screened from alpaca feces were isolated by Oxford Cup diffusion method and identified by 16S rRNA. The amino acid sequence of bacteriocins were obtained by (NH₄)₂SO₄ precipitation, chloroform extraction, SDS-PAGE and mass spectrometry analysis. The target bacteriocins were expressed by E. coli expression system, and the antibacterial activity and stability of the recombinant bacteriocins were identified by Oxford Cup diffusion. The results showed that the isolated strain was Bacillus subtilis, and was named as Bacillus subtilis SXAU18, the bacteriocins it produced could inhibit the growth of S.aureus, S. epidermidis, Micrococcus luteus and L. monocytogene. Analysis of the purified bacteriocins showed that they were probably a 10-20 ku DarA protein and two unknown proteins, which belonged to the bacteriocins category. The unknown proteins were named as BLIS SXAU181 and 182, respectively. Recombinant BLIS SXAU181 and 182 were mainly expressed in the soluble form in E. coli expression system, with a single band after purification. Recombinant BLIS SXAU182 had good antibacterial activity, and exhibited high resistance to high temperature, high acidity and alkalini, artificial gastri and intestinal fruid, but recombinant BLIS SXAU181 had no antibacterial activity. In conclusion, a bacteriocin with inhibitory activity against Gram-positive bacteria was isolated from Bacillus subtilis SXAU18 in this study, and the recombinant BLIS SXAU182 showed good antibacterial activity and stability.

Key words: Bacillus subtilis, bacteriocins, isolation, expression, stability

中图分类号:

S829.9

于秀菊, 张敏爱, 胡燕姣, 朱芷葳, 王海东, 杨丽华, 范阔海. 枯草芽孢杆菌细菌素的分离、表达及稳定性分析[J]. 畜牧兽医学报, 2024, 55(1): 323-333.

YU Xiuju, ZHANG Min'ai, HU Yanjiao, ZHU Zhiwei, WANG Haidong, YANG Lihua, FAN Kuohai. Isolation, Expression and Stability Analysis of Bacteriocins from Bacillus subtilis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 323-333.

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