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

Abstract

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

CLC Number:

S858.23

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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Construction of Brucella abortus A19 VirB Promoter Deletion Strain and Study on Its Biological Characteristics

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