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
Screening,validation,and functional analysis of Brucella secretory BspE interac-ting host proteins
印双红 1邓肖玉 2刘虹燕 1王海啸 2易彩霞 1李寅翠 1孙馨 1王书利 3易继海 2张俊波4
作者信息
- 1. 铜仁学院大健康学院,贵州铜仁 554300
- 2. 石河子大学动物科技学院,新疆石河子 832000
- 3. 商丘师范学院生物与食品学院,河南商丘 476000
- 4. 铜仁学院农林工程与规划学院,贵州铜仁 554300;铜仁学院贵州省梵净山地区生物多样性保护与利用重点实验室,贵州铜仁 554300
- 折叠
摘要
为了探究BspE蛋白在布鲁菌感染中的作用,利用酵母双杂交技术筛选与BspE蛋白互作的宿主细胞蛋白质.以构建的BspE重组质粒pGBKT7-BspE作为诱饵质粒,采用酵母双杂交技术,与小鼠单核巨噬细胞RAW264.7-cD-NA文库进行杂交.将获得的阳性克隆菌落经质粒抽提、测序分析和免疫共沉淀试验,确定可与BspE互作的宿主细胞蛋白.利用激光共聚焦显微镜分析BspE蛋白的亚细胞定位.利用生物信息学分析BspE互作蛋白的理化性质、蛋白结构、功能等信息.合成其中一个BspE互作蛋白的siRNA,沉默HEK293T细胞该蛋白基因的表达,将布鲁菌M5-90感染沉默的细胞,进行胞内细菌数量计数.结果显示,成功构建诱饵质粒pGBKT7-BspE,该质粒可在酵母菌中表达BspE蛋白.采用酵母双杂交技术从宿主细胞基因组文库获得8个阳性克隆,经测序、回交试验、免疫共沉淀验证后明确可与BspE蛋白互作的宿主蛋白为RBM27和PCBP1;利用生物信息学预测分析RBM27和PCBP1的细胞定位、蛋白结构及氨基酸种类组成;通过siRNA干扰后,PCBP1表达水平显著下降,胞内M5-90数量增加.研究表明,布鲁菌分泌蛋白BspE与宿主蛋白RBM27和PCBP1存在相互作用,且PCBP1对布鲁菌的增殖具有负调控作用.
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.
关键词
布鲁菌/酵母双杂交/免疫共沉淀/互作蛋白/生物信息学分析/RNA干扰Key words
Brucella/yeast two-hybrid/immunocoprecipitation/interacting protein/bioinformatics analysis/RNA interference引用本文复制引用
基金项目
国家自然科学基金(82160391)
国家自然科学基金(31502067)
贵州省科技计划(黔科合基础-ZK[2021]一般078)
铜仁市科技局项目(铜市科研[2022]67号)
铜仁市科技局项目(铜市科研[2023]39号)
贵州省高层次创新人才培养项目(2018-2016-022)
贵州省大学生创新创业训练计划(S202310665003)
贵州省大学生创新创业训练计划(S202310665004)
贵州省重点实验室项目(黔科合平台人才[2020]2003)
出版年
2024
NETL
NSTL
万方数据