牛种布鲁氏菌A19 VirB启动子缺失株的构建及其生物学特性研究

doi:10.16431/j.cnki.1671-7236.2020.03.026

摘要/Abstract

摘要： 为探究VirB基因对牛种布鲁氏菌A19株毒力的影响，深入了解布鲁氏菌胞内存活的机制，本研究以牛种布鲁氏菌A19株为模板，利用VirB基因的上下游同源臂融合kana抗性基因构建自杀质粒。以瞬间电击的方式，将自杀质粒电转进菌体，利用同源重组将VirB启动子用kana基因替换，构建A19ΔVirB缺失株。通过实时荧光定量PCR检测缺失株VirB相关蛋白的转录水平，并对缺失株的生长曲线、体外应激、黏附侵袭及胞内生存进行分析。结果显示，试验成功构建A19ΔVirB缺失株。实时荧光定量PCR结果显示，缺失株VirB相关蛋白的表达量极显著低于A19株（P<0.01），其生长曲线虽然不同于亲本株，但生长趋势相同。体外应激结果显示，A19株和A19ΔVirB株热休克应激没有明显变化，但在强酸、强碱、高盐的刺激下，A19株的存活率显著高于A19ΔVirB株（P<0.05）。黏附侵袭结果显示，缺失株对巨噬细胞的黏附侵袭能力近似于亲本株。胞内生存结果显示，随着时间的延长，A19株的胞内存活趋势逐渐上升，而缺失株A19ΔVirB总体趋势逐渐下降并且与A19的差距越来越大。综上所述，本研究成功构建了VirB启动子缺失株，极显著降低了相关蛋白的表达，为后续相关缺失株的构建及布鲁氏菌毒力的研究奠定基础。

关键词: 布鲁氏菌; A19ΔVirB; kana替换; 体外应激; 黏附侵袭; 胞内生存

Abstract: In order to explore the effects of VirB gene on the virulence of Brucella abortus A19,and learn more about the mechanism of Brucella intracellular survival,in this study,Brucella abortus A19 was used as a template to construct a suicide plasmid using the upstream and downstream homologous arms of VirB gene to fuse the kana resistance gene.The suicide plasmid was electroporated into the cells by means of an instantaneous electric shock,and the VirB promoter was replaced with kana gene by homologous recombination to construct an A19ΔVirB deletion strain.The transcription level of the VirB-associated protein of the deletion strain was detected by Real-time PCR,and the growth curve,in vitro stress,adhesion invasion,and intracellular survival of the deletion strain were analyzed.The results showed that the A19ΔVirB deletion strain was successfully constructed.Real-time PCR results showed that the expression of the VirB-associated protein of the deleted strain was extremely significantly lower than that of A19 strain (P<0.01).Although the growth curve was different from the parent strain,the growth trend was the same.The results of in vitro stress showed that there was no significant change in A19 and A19ΔVirB heat shock stress,but the survival rate of A19 was significantly higher than A19ΔVirB under the stimulation of strong acid,alkali and salt (P<0.05).The results of adhesion and invasion showed that the adhesion and invasion ability of the deletion strain to macrophages was similar to that of the parent strain.The results of intracellular survival showed that the intracellular survival trend of A19 gradually increased with time,while the overall trend of the deletion strain A19ΔVirB gradually decreased and the gap with A19 was larger.In summary,this study successfully constructed the VirB promoter deletion strain,which significantly reduced the expression of related proteins,and laid the foundation for the construction of subsequent related deletion strains and the study of Brucella virulence.

Key words: Brucella; A19ΔVirB; kana replacement; in vitro stress; adhesion invasion; intracellular survival

中图分类号:

S858.23

邓肖玉, 何金科, 杨琴, 易继海, 王月丽, 席静, 王震, 陈创夫. 牛种布鲁氏菌A19 VirB启动子缺失株的构建及其生物学特性研究[J]. 中国畜牧兽医, 2020, 47(3): 864-872.

DENG Xiaoyu, HE Jinke, YANG Qin, YI Jihai, WANG Yueli, XI Jing, WANG Zhen, CHEN Chuangfu. Construction of Brucella abortus A19 VirB Promoter Deletion Strain and Study on Its Biological Characteristics[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 864-872.

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