Effect of Brucella BspL Protein on the Expression Level of Inflammatory Factors in Macrophages and Bioinformatics Analysis

LI Zhiqiang; ZHU Fang; WANG Shuli; DING Jinkuo; DAI Yawen; ZHENG Hao; WANG Chengyu; LI Tianle

doi:10.15933/j.cnki.1004-3268.2025.06.016

Abstract：To explore the regulatory effect of Brucella secreted protein L(BspL)on the inflammatory response of macrophages,based on the BspL gene sequence(BAB1_1533)of Brucella abortus 2308 strain,an expression vector of pET-32a-BspL was constructed,and the BspL recombinant protein(rBspL)was expressed and purified.After identification and concentration determination by SDS-PAGE,rBspL was used to stimulate RAW264.7 macrophages.The mRNA expression levels of inflammatory factors(IL-1β and IL-18)and inflammasomes(NLRP3 and Caspase-1)were detected by qRT-PCR,and the protein expression levels of inflammasomes(NLRP3 and Caspase-1)were analyzed by Western blot.The bioinformatic analysis of BspL was performed using online softwares.The results showed that the pET-32a-BspL expression vector was successfully constructed,pET-32a-BspL expression product was purified to show a specific band at 36 ku.After stimulating RAW264.7 cells with rBspL,compared with the control group(DMEM was added),the experimental group(RAW264.7 cells were stimulated with 50 μg/mL rBspL)significantly increased the relative mRNA expression levels of NLRP3,Caspase-1,IL-1β and IL-18 and the protein expression levels of inflammasomes NLRP3 and Caspase-1 were significantly increased.Bioinformatics analysis showed that BspL protein was hydrophilic,with two transmembrane regions,two secretion pathway signal peptides(Sec signal peptides),26 phosphorylation sites and five antigenic epitopes.The secondary structure of BspL protein was dominated by random coil and α-helix,and there were a few extended chains.In addition,the results of tertiary structure prediction showed that most of the BspL protein structures were random coil and α-helix,which was consistent with the results of secondary structure prediction.In conclusion,Brucella rBspL expressed by Escherichia coli could induce host cells to release inflammatory factors,which provided references for further study of the function and molecular mechanism of BspL.