核效应蛋白BspG、BspJ在布鲁氏菌胞内生存的功能与调节机制探索

布鲁氏菌效应蛋白在调节其胞内生存中发挥重要作用，但其功能与调节机制仍需深入研究。前期研究证明：布鲁氏菌BspG、BspJ能够定位于宿主细胞核，是核效应蛋白；据此我们提出一个科学假设：核效应蛋白BspG、BspJ可能是一个转录调节因子，通过与宿主的DNA和/或蛋白质相互作用，影响宿主细胞功能，调节布鲁氏菌胞内生存。为验证该科学假设，本研究使用ChIP-seq、酵母双杂交（YTH）分别筛选与BspG和BspJ互作的靶标DNA和/或蛋白质；使用凝胶电泳迁移率（EMSA）、Co-IP、RNAi等方法验证与BspG和BspJ互作的靶标；同源重组分析核效应蛋白BspG和BspJ缺失株毒力表型；探究BspG、BspJ调节布鲁氏菌胞内生存的功能及分子机制。本研究的开展及实验结果，将为解析布鲁氏菌胞内生存分子机制提供新理论依据。

The effectors from Brucella plays pivotal roles in regulating intracellular survival, However, the functions and regulatory mechanisms of effectors in mediating Brucella intracellular growth needs further research. Our previous researchs suggested that BspG and BspJ were localized in the nucleus of host cells and may be nuclear-cytoplasm shuttle effector proteins (designated as nucleomodulins). Based on the above observation, we proposed the hypothesis that nucleomodulins BspG and BspJ may be transcription regulators, which regulate host functions and Brucella intracellular survival through DNA-protein and/or protein-protein interaction. To prove the hypothesis, ChIP-seq and Yeast Two Hybrid (YTH) will be used to screen targets DNA and/or protiens interaction with nucleomodulins BspG and BspJ; Some methods including Electrophoretic Mobility Shift Assay (EMSA)、Co-Immunoprecipitation (CO-IP) and RNA interference (RNAi), etc will be used to elucidate that nucleomodulins BspG and BspJ may interact with host targets DNA and/or proteins；our team in the work will analyze the virulence phenontypes of nucleomodulins BspG and BspJ mutants by homologous recombination. In addition, the molecular regulatory mechanism by which the BspG and BspJ regulate Brucella intracellular growth will be studied in this project..If the proposal is funded, we will explore functions and regulation mechanismsof nucleomodulins for the first time, our studies will potentially give an insight into how the nucleomodulins regulate Brucella intracellular survival.