Screening,validation,and functional analysis of Brucella secretory BspE interac-ting host proteins
10.16303/j.cnki.1005-4545.2024.07.13
- VernacularTitle:布鲁菌分泌蛋白BspE互作的宿主蛋白的筛选、验证及功能分析
- Author:
Shuanghong YIN
1
;
Xiaoyu DENG
;
Hongyan LIU
;
Haixiao WANG
;
Caixia YI
;
Yincui LI
;
Xin SUN
;
Shuli WANG
;
Jihai YI
;
Junbo ZHANG
Author Information
1. 铜仁学院大健康学院,贵州铜仁 554300
- Keywords:
Brucella;
yeast two-hybrid;
immunocoprecipitation;
interacting protein;
bioinformatics analysis;
RNA interference
- From:
Chinese Journal of Veterinary Science
2024;44(7):1438-1447,1457
- CountryChina
- Language:Chinese
-
Abstract:
In order to explore the role of BspE protein in Brucella infection,yeast two-hybrid tech-nique was used to screen host cell proteins that interact with BspE protein.The constructed BspE recombinant plasmid pGBKT7-BspE was used as bait plasmid to hybridize with the RAW264.7-cD-NA library of mouse mononuclear macrophages by yeast two-hybridization technique.The positive clones were extracted by plasmid,sequenced and co-immunoprecipitation to determine the host cell proteins that could interact with BspE.The subcellular localization of BspE proteins was analyzed by confocal laser microscopy.The physical and chemical properties,protein structure and function of BspE interacting proteins were analyzed by bioinformatics.The siRNA for one of the BspE inter-acting proteins was synthesized,the expression of its gene was silenced in HEK293T cells,and the silenced cells was infected with Brucella M5-90 and the number of intracellular bacteria was coun-ted.The results showed that the decoy plasmid pGBKT7-BspE was successfully constructed,and the plasmid could express BspE protein in yeast.Eight positive clones were obtained from the host cell genome library by yeast two-hybridization.The positive clones were identified as RBM27 and PCBP1 by sequencing,backcross and co-immunoprecipitation.Bioinformatics was used to predict the cell location,protein structure and amino acid composition of RBM27 and PCBP1.After siRNA interference,the expression level of PCBP1 was significantly decreased and the amount of M5-90 in the cell was increased.Brucellosis secreted protein BspE interacts with host proteins RBM27 and PCBPl,and PCBP1 negatively regulates the proliferation of Brucellosis.
