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

Abstract

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

CLC Number:

S852.61⁺4

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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Subcellular Localization and Screening of Host Interacting Proteins of Brucella Secreted Protein BPE005

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