布鲁氏菌分泌蛋白BPE005的亚细胞定位及与宿主细胞互作蛋白的筛选

doi:10.16431/j.cnki.1671-7236.2022.04.026

摘要/Abstract

摘要： 【目的】分析布鲁氏菌分泌蛋白BPE005的蛋白结构及细胞定位,筛选宿主细胞内与其存在相互作用的靶蛋白,为后续研究BPE005的生物学功能奠定基础。【方法】通过生物信息学软件对BPE0005的蛋白结构进行初步分析;构建PDRRED2-C1-BPE005重组荧光定位载体,转染人胚肾上皮细胞293T,通过扫描激光共聚焦显微镜观察BPE005的细胞定位;构建PGBKT7-BPE005诱饵载体,验证诱饵载体是否有细胞毒性和自激活现象;以小鼠巨噬细胞RAW264.7 mRNA的cDNA文库为AD文库菌液,检测文库滴度,通过酵母双杂交系统筛选宿主细胞内与BPE005相互作用的靶蛋白。【结果】布鲁氏菌分泌蛋白BPE005属于疏水性蛋白,内含有大量的无规则卷曲、α-螺旋和延伸链。成功构建PDRRED2-C1-BPE005荧光定位载体和PGBKT7-BPE005诱饵蛋白载体,BPE005定位于宿主细胞核。PGBKT7-BPE005诱饵蛋白载体无酵母细胞毒性和自激活现象,AD文库菌液滴度>2×10⁷/mL,筛选出4个宿主细胞内与BPE0005存在相互作用的蛋白:RNA聚合酶Ⅱ多肽G、补体因子H、鸟苷酸环化酶2G及颗粒蛋白。经复筛、测序、BLAST比对、一对一验证后,最终筛选出1个在宿主细胞内与BPE005具有较强相互作用的靶蛋白(RNA聚合酶Ⅱ多肽G)。对该蛋白的作用网络分析表明,RNA聚合酶Ⅱ多肽G对宿主细胞内mRNA的转录和合成具有重大影响。【结论】布鲁氏菌分泌蛋白BPE005定位于宿主细胞核,在宿主细胞内可与RNA聚合酶Ⅱ多肽G产生较强相互作用,对进一步阐明布鲁氏菌感染过程中关键效应分子的作用机制具有指导意义。

关键词: 布鲁氏菌; BPE005; 亚细胞定位; 酵母双杂交

Abstract: 【Objective】 This study was aimed to analyze the protein structure and cellular localization of Brucella secretory protein BPE005,and screen the target proteins that interact with it in host cells,so as to lay a foundation for the follow-up study of the biological function of BPE005.【Method】 The protein structure of BPE0005 was analyzed by bioinformatics software,the recombinant fluorescent localization vector of PDRRED2-C1-BPE005 was constructed and transfected into human renal epithelial cells 293T,the cellular localization of BPE005 was observed by scanning laser confocal microscope.The PGBKT7-BPE005 bait vector was constructed to verify whether the bait vector had cytotoxicity and self-activation.The cDNA library of mouse macrophage RAW264.7 mRNA was used as AD library,and the titer of the library was detected.The target proteins interacting with BPE005 in host cells were screened by yeast two-hybrid system.【Result】 Brucella secretory protein BPE005 was hydrophobic and contained a large number of random coil,alpha helix and extended chain.The PDRRED2-C1-BPE005 fluorescence localization vector and PGBKT7-BPE005 bait protein vector were successfully constructed.BPE005 was located in the host nucleus.The PGBKT7-BPE005 bait protein vector had no yeast cytotoxicity and self-activation.The titer of AD library was more than 2×10⁷/mL.Four host cell proteins interacting with BPE0005,RNA polymerase Ⅱ polypeptide G,complement factor H,guanylatecyclase 2G and granule protein were screened.After rescreening,sequencing,BLAST comparison and one-on-one verification,a target protein (RNA polymerase Ⅱ peptide G) with strong interaction with BPE005 in host cells was selected.The analysis of the action network of the protein showed that RNA polymerase Ⅱ peptide G had a significant effect on the transcription and synthesis of mRNA in host cells.【Conclusion】 Brucella secretory protein BPE005 was located in the host nucleus,and could interact strongly with polypeptide G of RNA polymerase Ⅱ in host cells,which was of guiding significance for further elucidating the action mechanism of key effector molecules in the process of Brucella infection.

Key words: Brucella; BPE005; subcellular localization; yeast two-hybrid

中图分类号:

S852.61⁺4

邱润辉, 关飞虎, 陶婷婷, 李佳, 赵天艺, 邓兴梅, 史超, 孙志华, 张辉. 布鲁氏菌分泌蛋白BPE005的亚细胞定位及与宿主细胞互作蛋白的筛选[J]. 中国畜牧兽医, 2022, 49(4): 1438-1448.

QIU Runhui, GUAN Feihu, TAO Tingting, LI Jia, ZHAO Tianyi, DENG Xingmei, SHI Chao, SUN Zhihua, ZHANG Hui. Subcellular Localization and Screening of Host Interacting Proteins of Brucella Secreted Protein BPE005[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1438-1448.

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